U.S. patent application number 16/171837 was filed with the patent office on 2019-07-18 for methods and compositions for preserving photoreceptor and retinal pigment epithelial cells.
The applicant listed for this patent is Massachusetts Eye and Ear Infirmary. Invention is credited to Joan W. Miller, Yusuke Murakami, Georgios Trichonas, Demetrios G. Vavvas.
Application Number | 20190216881 16/171837 |
Document ID | / |
Family ID | 44627940 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190216881 |
Kind Code |
A1 |
Vavvas; Demetrios G. ; et
al. |
July 18, 2019 |
METHODS AND COMPOSITIONS FOR PRESERVING PHOTORECEPTOR AND RETINAL
PIGMENT EPITHELIAL CELLS
Abstract
Provided are methods and compositions for maintaining the
viability of photoreceptor cells and/or retinal pigment epithelial
cells in a subject with an ocular disorder including, for example,
age-related macular degeneration (AMD) (e.g., dry or neovascular
AMD), retinitis pigmentosa (RP), or a retinal detachment. The
viability of the photoreceptor cells and/or the retinal pigment
epithelial cells can be preserved by administering a necrosis
inhibitor either alone or in combination with an apoptosis
inhibitor to a subject having an eye with the ocular condition. The
compositions, when administered, maintain the viability of the
cells, thereby minimizing the loss of vision or visual function
associated with the ocular disorder.
Inventors: |
Vavvas; Demetrios G.;
(Boston, MA) ; Trichonas; Georgios; (Boston,
MA) ; Miller; Joan W.; (Winchester, MA) ;
Murakami; Yusuke; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Eye and Ear Infirmary |
Boston |
MA |
US |
|
|
Family ID: |
44627940 |
Appl. No.: |
16/171837 |
Filed: |
October 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15288214 |
Oct 7, 2016 |
10149884 |
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16171837 |
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14934810 |
Nov 6, 2015 |
9492432 |
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15288214 |
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13642887 |
Feb 14, 2013 |
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PCT/US2011/033704 |
Apr 23, 2011 |
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14934810 |
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61472153 |
Apr 5, 2011 |
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61472144 |
Apr 5, 2011 |
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61414862 |
Nov 17, 2010 |
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61409055 |
Nov 1, 2010 |
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61405003 |
Oct 20, 2010 |
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61327476 |
Apr 23, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
A61F 9/0008 20130101; A61K 31/416 20130101; A61K 31/4178 20130101;
A61K 31/4174 20130101; A61K 31/12 20130101; A61B 3/028 20130101;
A61K 38/05 20130101; A61K 45/06 20130101; A61K 38/06 20130101; A61K
38/005 20130101; A61K 31/195 20130101; A61K 9/0048 20130101; A61B
5/4839 20130101; A61P 27/02 20180101; A61K 31/4155 20130101; A61K
31/40 20130101; A61K 31/4174 20130101; A61K 2300/00 20130101; A61K
31/195 20130101; A61K 2300/00 20130101; A61K 31/4178 20130101; A61K
2300/00 20130101; A61K 31/12 20130101; A61K 2300/00 20130101; A61K
31/40 20130101; A61K 2300/00 20130101; A61K 31/519 20130101; A61K
2300/00 20130101; A61K 31/4155 20130101; A61K 2300/00 20130101;
A61K 31/416 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/05 20060101
A61K038/05; A61K 38/00 20060101 A61K038/00; A61K 31/4178 20060101
A61K031/4178; A61K 31/519 20060101 A61K031/519; A61K 31/4174
20060101 A61K031/4174; A61K 31/416 20060101 A61K031/416; A61K
31/4155 20060101 A61K031/4155; A61K 31/40 20060101 A61K031/40; A61K
31/195 20060101 A61K031/195; A61K 31/12 20060101 A61K031/12; A61F
9/00 20060101 A61F009/00; A61B 3/028 20060101 A61B003/028; A61B
5/00 20060101 A61B005/00; A61K 38/06 20060101 A61K038/06; A61K 9/00
20060101 A61K009/00; A61K 45/06 20060101 A61K045/06 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The work described in this application was sponsored, in
part, by the National Eye Institute under Grant No. EY14104. The
United States Government has certain rights in the invention.
Claims
1-56. (canceled)
57. A composition for preserving the viability of photoreceptor
cells, preserving the viability of retinal pigment epithelial
cells, or preserving or improving visual function in an eye of a
subject with retinal detachment, the composition comprising: a
pharmaceutically acceptable carrier, a necrosis inhibitor and an
apoptosis inhibitor.
58. The composition of claim 57, wherein the necrosis inhibitor is
a necrostatin selected from the group consisting of necrostatin-1,
necrostatin-2, necrostatin-3, necrostatin-4, necrostatin-5, and
necrostatin-7, or a combination thereof.
59. The composition of claim 58, wherein the necrostatin is an
(R)-enantiomer of necrostatin-1.
60. The composition of claim 57, wherein the apoptosis inhibitor is
a pan-caspase inhibitor.
61. The composition of claim 60, wherein the pan-caspase inhibitor
is selected from the group consisting of Z-VAD, IDN-6556, and a
combination of Z-VAD and IDN-6556.
62. The composition of claim 57, wherein the necrosis inhibitor,
the apoptosis inhibitor, or both the necrosis inhibitor and the
apoptosis inhibitor are formulated for intravitreal or intraocular
administration.
63. The composition of claim 57, wherein the necrosis inhibitor,
the apoptosis inhibitor, or both the necrosis inhibitor and the
apoptosis inhibitor are formulated for sequential or simultaneous
administration.
64. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-1 related compound of Formula I: ##STR00068## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: X is O or S; R.sub.1 is hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxyl, or halogen; and R.sub.2 is hydrogen or
C.sub.1-C.sub.6alkyl.
65. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-1 compound of Formula I-A: ##STR00069## or a pharmaceutically
acceptable salt, ester, or prodrug thereof, wherein: R.sub.1 is H,
alkyl, alkoxyl, or a halogen; and R.sub.2 is H or an alkyl.
66. The composition of claim 57, wherein the necrosis inhibitor is
selected from the group consisting of: a Nec-1 related compound
Formula I-B: ##STR00070## or a pharmaceutically acceptable salt,
ester, or prodrug thereof; a Nec-1 related compound of Formula I-C:
##STR00071## or a pharmaceutically acceptable salt, ester, or
prodrug thereof, and a Nec-1 related compound of Formula I-D:
##STR00072## or a pharmaceutically acceptable salt thereof.
67. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-1 related compound of Formula I-E: ##STR00073## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein R.sub.1 is H, alkyl, alkoxyl, or a halogen; and R.sub.2 is
H or an alkyl.
68. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-2 related compound of Formula II: ##STR00074## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: X is --CH.sub.2--, --C(H)(R.sub.14)--, --C(.dbd.S)--,
--C(.dbd.NH)--, or --C(O)--; R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10 each
represent independently hydrogen, acyl, acetyl, alkyl, halogen,
amino, C.sub.1-C.sub.6alkoxyl, nitro, --C(O)R.sub.12,
--C(S)R.sub.12, --C(O)OR.sub.12, --C(O)NR.sub.12R.sub.13,
--C(S)NR.sub.12R.sub.13, or --S(O.sub.2)R.sub.12; R.sub.11 is
hydrogen, acyl, acetyl, alkyl, or acylamino; R.sub.12 and R.sub.13
each represent independently hydrogen, an optionally substituted
alkyl, an optionally substituted aryl, an optionally substituted
heteroaryl, an optionally substituted aralkyl, or an optionally
substituted heteroaralkyl; R.sub.14 is acyl, acetyl, alkyl,
halogen, amino, acylamino, nitro, --SR.sub.11, --N(R.sub.11).sub.2,
or --OR.sub.11; the bond indicated by (a) can be a single or double
bond; and the bond indicated by (b) can be a single or double
bond.
69. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-2 related compound of Formula II-A: ##STR00075## or a
pharmaceutically acceptable salt thereof, wherein: R.sub.1,
R.sub.2, R.sub.5, R.sub.6, R.sub.7, and R.sub.10 each represent
independently hydrogen, alkyl, halogen, amino, or methoxyl; and
R.sub.3, R.sub.4, R.sub.8, and R.sub.9 are C.sub.1-C.sub.6
alkoxyl.
70. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-3 related compound of Formula III: ##STR00076## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: Z is --CH.sub.2--, --CH.sub.2CH.sub.2--, --O--, --S--,
--S(O)--, --S(O.sub.2)--, or --N(R.sub.7)--; R.sub.1, R.sub.3, and
R.sub.5 each represent independently for each occurrence hydrogen,
halogen, hydroxyl, amino, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxy, C.sub.1-C.sub.6alkoxy-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfinyl,
C.sub.1-C.sub.6alkylsulfinyl-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonyl-C.sub.1-C.sub.6alkyl, aryl, aralkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl; R.sub.2 and R.sub.4
are C.sub.1-C.sub.6alkoxy; R.sub.6 is --C(O)R.sub.8, --C(S)R.sub.8,
--C(O)OR.sub.8, --C(O)NR.sub.8R.sub.9, --C(S)NR.sub.8R.sub.9,
--C(NH)R.sub.8, or --S(O.sub.2)R.sub.8; R.sub.7 is alkyl, aralkyl,
or heteroaralkyl; R.sub.8 and R.sub.9 each represent independently
hydrogen, C.sub.1-C.sub.6alkyl, heteroalkyl, aryl, heteroaryl,
aralkyl, or heteroaralkyl; and n represents independently for each
occurrence 0, 1, or 2.
71. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-4 related compound of Formula IV: ##STR00077## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: R.sub.1 is ##STR00078## R.sub.2 and R.sub.3 each represent
independently for each occurrence hydrogen or methyl; R.sub.4
represents independently for each occurrence halogen, hydrogen,
C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl, or
C.sub.2-C.sub.4alkynyl; R.sub.5 is C.sub.1-C.sub.4alkyl; R.sub.6 is
hydrogen, halogen, or --CN; R.sub.7 is hydrogen or
C.sub.1-C.sub.4alkyl; R.sub.8 is C.sub.1-C.sub.6alkyl, or R.sub.8
taken together with R.sub.9, when present, forms a carbocyclic
ring; R.sub.9 is hydrogen or C.sub.1-C.sub.6alkyl, or R.sub.9 taken
together with R.sub.8 forms a carbocyclic ring; R.sub.10 is
hydrogen or C.sub.1-C.sub.6alkyl; A is phenylene or a 5-6 membered
heteroarylene; X is N or --C(R.sub.9)--; Y is N or --C(R.sub.10)--;
Z is S or O; and m and n each represent independently 1, 2, or
3.
72. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-5 related compound of Formula V: ##STR00079## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: A is a saturated or unsaturated 5-6 membered carbocyclic
ring; X is a bond or C.sub.1-C.sub.4alkylene; R.sub.1 is
C.sub.1-C.sub.6 alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2; R.sub.2 is ##STR00080## R.sub.3 is
--C.sub.1-C.sub.6alkylene-CN, --CN, C.sub.1-C.sub.6alkyl, or
C.sub.2-C.sub.6alkenyl; R.sub.4 represents independently for each
occurrence hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl;
R.sub.5 represents independently for each occurrence
C.sub.1-C.sub.6alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2; B is a 5-6 membered heterocyclic or
carbocylic ring; and n and p each represent independently 0, 1, or
2.
73. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-5 related compound of Formula V-A: ##STR00081## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: R.sub.1 is C.sub.1-C.sub.6alkyl, halogen, hydroxyl,
C.sub.1-C.sub.6alkoxyl, or --N(R.sub.4).sub.2; R.sub.2 is
##STR00082## R.sub.3 is --C.sub.1-C.sub.6alkylene-CN; R.sub.4
represents independently for each occurrence hydrogen,
C.sub.1-C.sub.6alkyl, aryl, or aralkyl; R.sub.5 represents
independently for each occurrence C.sub.1-C.sub.6alkyl, halogen,
hydroxyl, C.sub.1-C.sub.6alkoxyl, --N(R.sub.4).sub.2,
--C(O)R.sub.4, CO.sub.2R.sub.4, or C(O)N(R.sub.4).sub.2; B is a 5-6
membered heterocyclic or carbocylic ring; and n and p each
represent independently 0, 1, or 2.
74. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-7 related compound of Formula VII: ##STR00083## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: R.sub.1, R.sub.2, and R.sub.3 each represent independently
hydrogen or C.sub.1-C.sub.4alkyl; R.sub.4 is ##STR00084## R.sub.5
and R.sub.6 each represent independently for each occurrence
halogen, C.sub.1-C.sub.6alkyl, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.7).sub.2, --NO.sub.2, --S--C.sub.1-C.sub.6alkyl,
--S-aryl, --SO.sub.2--C.sub.1-C.sub.6alkyl, --SO.sub.2-aryl,
--C(O)R.sub.7, --CO.sub.2R.sub.7, --C(O)N(R.sub.7).sub.2,
heterocycloalkyl, aryl, or heteroaryl; R.sub.7 represents
independently for each occurrence hydrogen, C.sub.1-C.sub.6alkyl,
aryl, or aralkyl; or two occurrences of R.sub.7 attached to the
same nitrogen atom are taken together with the nitrogen atom to
which they are attached to form a 3-7 membered heterocyclic ring; A
is a 5-6 membered heterocyclic ring; and p is 0, 1, or 2.
75. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-8 related compound of Formula VIII: ##STR00085## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: each X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 is selected, independently, from N or CR.sup.X1; each
Y.sup.1, Y.sup.2, and Y.sup.3 is selected, independently, from O,
S, NR.sup.Y1, or CR.sup.Y2R.sup.Y3; each Z.sup.1 and Z.sup.2 is
selected, independently, from O, S, or NR.sup.Z1; each R.sup.Y1 and
R.sup.Z1 is selected, independently, from H, optionally substituted
C.sub.1-6alkyl, optionally substituted C.sub.2-6alkenyl, optionally
substituted C.sub.2-6alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, --C(.dbd.O)R.sup.5A,
--C(.dbd.O)OR.sup.5A, or --C(.dbd.O)NR.sup.5AR.sup.6A; each
R.sup.X1, R.sup.Y2, and R.sup.Y3 is selected, independently, from
H, halogen, CN, NC, NO.sub.2, N.sub.3, OR.sup.3, SR.sup.3,
NR.sup.3R.sup.4, --C(.dbd.O)R.sup.5A, --C(.dbd.O)OR.sup.5A,
--C(.dbd.O)NR.sup.5AR.sup.6A, --S(.dbd.O)R.sup.5A,
--S(.dbd.O).sub.2R.sup.5A, --S(.dbd.O).sub.2OR.sup.5A,
--S(.dbd.O).sub.2NR.sup.5AR.sup.6A, optionally substituted
C.sub.1-6alkyl, optionally substituted C.sub.2-6alkenyl, optionally
substituted C.sub.2-6alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
or optionally substituted heteroaryl; each R.sup.1, R.sup.2
R.sup.5A, R.sup.5B, R.sup.6A, and R.sup.6B is selected from H,
optionally substituted C.sub.1-6 alkyl, optionally substituted
C.sub.2-6alkenyl, optionally substituted C.sub.2-6alkynyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, or optionally
substituted heteroaryl; or R.sup.5A and R.sup.6A, or R.sup.5B and
R.sup.6B combine to form a heterocyclyl; and each R.sup.3 and
R.sup.4 is selected from H, optionally substituted C.sub.1-6 alkyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, --C(.dbd.O)R.sup.5B, --C(.dbd.S)R.sup.5B,
--C(.dbd.NR.sup.6B)R.sup.5B, --C(.dbd.O)OR.sup.5B,
--C(.dbd.O)NR.sup.5BR.sup.6B, --S(.dbd.O)R.sup.5B,
--S(.dbd.O).sub.2R.sup.5B, --S(.dbd.O).sub.2OR.sup.5B, or
--S(.dbd.O).sub.2NR.sup.5BR.sup.6B.
76. The composition of claim 57, wherein the necrosis inhibitor is
a Nec-4 related compound of Formula IX: ##STR00086## or a
pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein: X.sub.1 and X.sub.2 are, independently, N or CR.sup.4;
X.sub.3 is selected from O, S, NR.sup.5, or --(CR.sup.5).sub.2; Y
is selected from C(O) or CH.sub.2; and Z is
(CR.sup.6R.sup.7).sub.n; R.sup.1 is selected from H, halogen,
optionally substituted C.sub.1-6alkyl, or optionally substituted
C.sub.1-6 cycloalkyl, or optionally substituted aryl; R.sup.2 is
selected from H or optionally substituted C.sub.1-6alkyl; R.sup.3
is optionally substituted aryl; each R.sup.4 is selected from H,
halogen, carboxamido, nitro, cyano, optionally substituted
C.sub.1-6 alkyl, or optionally substituted aryl; R.sup.5 is
selected from H, halogen, optionally substituted C.sub.1-6alkyl, or
optionally substituted aryl; each R.sup.6 and R.sup.7 is,
independently, selected from H, optionally substituted
C.sub.1-6alkyl, or aryl; and n is 0, 1, 2, or 3.
77. A pharmaceutical composition formulated for intraocular
administration to an eye of a subject with an ocular condition, the
composition comprising a pharmaceutically acceptable carrier, a
necrosis inhibitor, and an apoptosis inhibitor, wherein the
composition is formulated to provide a final concentration of the
necrosis inhibitor in the eye in the range from about 150 .mu.M to
about 1000 .mu.M, and wherein the composition is formulated to
provide a final concentration of the apoptosis inhibitor in the
range from about 150 .mu.M to about 500 .mu.M.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/288,214, filed Oct. 7, 2016, which is a
continuation of U.S. patent application Ser. No. 14/934,810, filed
Nov. 6, 2015 (and issued as U.S. Pat. No. 9,492,432 on Nov. 15,
2016), which is a continuation of U.S. patent application Ser. No.
13/642,887, filed Feb. 14, 2013 (now abandoned), which is the
national stage of International (PCT) Patent Application No.
PCT/US2011/033704, filed Apr. 23, 2011, which claims the benefit of
and priority to U.S. Provisional Application No. 61/327,476, filed
Apr. 23, 2010; U.S. Provisional Application No. 61/405,003, filed
Oct. 20, 2010; U.S. Provisional Application No. 61/409,055, filed
Nov. 1, 2010; U.S. Provisional Application No. 61/414,862, filed
Nov. 17, 2010; U.S. Provisional Application No. 61/472,153, filed
Apr. 5, 2011; and U.S. Provisional Application No. 61/472,144,
filed Apr. 5, 2011, the disclosure of each of which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The field of the invention relates generally to methods and
compositions for preserving the viability of photoreceptor cells
and/or retinal pigment epithelial cells, for example, in a subject
affected with an ocular condition wherein a symptom of the ocular
condition is loss of photoreceptor cell viability and/or retinal
pigment epithelial cell viability, e.g., age-related macular
degeneration (AMD), retinitis pigmentosa (RP), or a retinal
detachment. More particularly, the invention relates to the use of
a necrosis inhibitor, e.g., a RIP kinase inhibitor, e.g., a
necrostatin, either alone or in combination with an apoptosis
inhibitor, e.g., a pan-caspase inhibitor, for preserving the
viability of photoreceptor and/or retinal pigment epithelial cells
during the treatment of the ocular disorder.
BACKGROUND OF THE INVENTION
[0004] The retina is a delicate neural tissue lining the back of
the eye that converts light stimuli into electric signals for
processing by the brain. Ocular disorders affecting the retina,
including, for example, retinal detachment, AMD, and RP can lead to
vision loss and blindness. Early detection and treatment are
critical in correcting problems before vision is lost or in
preventing further deterioration of vision.
[0005] Photoreceptor death after retinal detachment ("RD") is a
major cause of permanent visual loss in various ocular diseases.
During retinal detachment, the entire retina or a portion of the
retina becomes dissociated from the underlying retinal pigment
epithelium and choroid. As a result, the sensitive photoreceptor
cells disposed in the detached portion of the retina become
deprived of their normal supply of blood and nutrients. If
untreated, the retina or, more particularly, the sensitive
photoreceptor cells disposed within the retina die causing partial
or even complete blindness. Physical separation of photoreceptors
from the underlying retinal pigment epithelium occurs in
age-related macular degeneration (Dunaief J L et al. (2002) ARCH.
OPHTHALMOL. 120:1435-1442), diabetic retinopathy (Barber A J et al.
(1998) J. CLIN. INVEST. 102:783-791), retinopathy of prematurity
(Fulton A B et al. (2001) ARCH. OPHTHALMOL. 119:499-505), as well
as rhegmatogenous (caused by a break in the retina), serous or
tractional retinal detachment (Cook B et al. (1995) INVEST.
OPHTHALMOL. VIS. SCI. 36:990-996; Arroyo et al. (2005) AM. J.
OPHTHALMOL. 139:605-610). Although surgery may be carried out to
reattach the retina, only two-fifths of patients with retinal
detachment involving the macula, a region essential for central
vision, recover 20/40 or better vision due to photoreceptor death
(Arroyo et al. (2005) AM. J. OPHTHALMOL. 139:605-610; Campo et al.
(1999) OPHTHALMOLOGY 106:1811-1815). Identification of the
mechanisms that underlie photoreceptor death is critical to
developing new treatment strategies for these diseases.
[0006] Age-related macular degeneration is the leading cause of
irreversible vision loss in the developed world affecting
approximately 15% of individuals over the age of 60. Macular
degeneration is categorized as either dry (atrophic) or wet
(neovascular). The dry form is more common than the wet, with about
90% of AMD patients diagnosed with dry AMD. In the dry form, there
is a breakdown or thinning of the retinal pigment epithelial cells
(RPE) in the macula, hence the term "atrophy." These RPE cells are
important to the function of the retina, as they metabolically
support the overlying photoreceptors. AMD is a challenging disease
for both patients and doctors because there are very few treatment
options. Current therapies, including laser photocoagulation,
photodynamic therapy, and anti-angiogenic therapeutics have had
mixed results, and, in certain instances, have caused deleterious
side effects. A need therefore exists for a treatment that reduces
or limits the effects of macular degeneration.
[0007] Retinitis pigmentosa (RP) is a group of genetic eye
conditions that leads to incurable blindness. Though the majority
of mutations target photoreceptors, some affect RPE cells directly.
Together, these mutations affect such processes as molecular
trafficking between photoreceptors and RPE cells and
phototransduction, for example. Currently, there is no cure for
RP.
[0008] Apoptosis and necrosis represent two different mechanisms of
cell death. Apoptosis is a highly regulated process involving the
caspase family of cysteine proteases, and characterized by cellular
shrinkage, chromatin condensation, and DNA degradation. In
contrast, necrosis is associated with cellular and organelle
swelling and plasma membrane rupture with ensuing release of
intracellular contents and secondary inflammation (Kroemer G et al.
(2009) CELL DEATH DIFFER. 16:3-11). Necrosis has been considered a
passive, unregulated form of cell death; however, recent evidence
indicates that some necrosis can be induced by regulated signal
transduction pathways such as those mediated by receptor
interacting protein (RIP) kinases, especially in conditions where
caspases are inhibited or cannot be activated efficiently (Golstein
P & Kroemer G (2007) TRENDS BIOCHEM. SCI. 32:37-43; Festjens N
et al. (2006) BIOCHIM. BIOPHYS. ACTA 1757:1371-1387). Stimulation
of the Fas and TNFR family of death domain receptors (DRs) is known
to mediate apoptosis in most cell types through the activation of
the extrinsic caspase pathway. In addition, in certain cells
deficient for caspase-8 or treated with pan-caspase inhibitor
Z-VAD, stimulation of death domain receptors (DR) causes a RIP-1
kinase dependent programmed necrotic cell death instead of
apoptosis (Holler N et al. (2000) NAT. IMMUNOL. 1:489-495; Degterev
A et al. (2008) NAT. CHEM. BIOL. 4:313-321). This novel mechanism
of cell death is termed "programmed necrosis" or "necroptosis"
(Degterev A et al. (2005) NAT. CHEM. BIOL. 1:112-119).
[0009] Receptor Interacting Protein kinase 1 (RIP-1) is a
serine/threonine kinase that contains a death domain and forms a
death signaling complex with the Fas-associated death domain and
caspase-8 in response to death receptor (DR) stimulation (Festjens
N et al. (2007) CELL DEATH DIFFER. 14:400-410). During death domain
receptor-induced apoptosis, RIP-1 is cleaved and inactivated by
caspase-8, the process of which is prevented by caspase inhibition
(Lin Y et al. (1999) GENES. DEV. 13:2514-2526). It has been unclear
how RIP-1 kinase mediates programmed necrosis, but recent studies
revealed that the expression of RIP-3 and the RIP-I1-RIP-3 binding
through the RIP homotypic interaction motif is a prerequisite for
RIP-1 kinase activation, leading to reactive oxygen species (ROS)
production and necrotic cell death (He S et al. (2009) CELL
137:1100-1111; Cho Y S et al. (2009) CELL 137:1112-1123; Zhang D W
et al. (2009) SCIENCE 325:332-336).
[0010] There is still an ongoing need to minimize or eliminate
photoreceptor and/or retinal pigment epithelial cell death in
certain ocular disorders, e.g., in AMD, RP, and retinal detachment.
It is contemplated that minimizing photoreceptor and/or retinal
pigment epithelial cell death will reduce the loss of vision or the
loss of visual function associated with these various
disorders.
SUMMARY OF THE INVENTION
[0011] The invention is based, in part, on the discovery that a
necrosis inhibitor, e.g., RIP kinase inhibitor, e.g., a
necrostatin, e.g., necrostatin-1, can be used to reduce or prevent
the loss of photoreceptor and/or retinal pigment epithelial cell
viability, especially when the necrosis inhibitor is combined with
an apoptotic inhibitor (e.g., a pan-caspase inhibitor, e.g., Z-VAD
and/or IDN-6556). It was previously understood that photoreceptor
cell death associated with AMD, RP, and retinal detachment was
primarily caused by apoptosis. However, studies have shown that the
administration of Z-VAD, a pan-caspase inhibitor, fails to prevent
photoreceptor loss in these conditions. The studies described
hereinbelow indicate that, in the presence of an apoptosis
inhibitor (e.g., a pan-caspase inhibitor), photoreceptors die by
necrosis, including necroptosis (or programmed necrosis). These
studies show that programmed necrosis is a critical mechanism for
ocular conditions wherein a symptom of the condition is the loss of
photoreceptor cell viability in the presence of a pan-caspase
inhibitor. As a result, it is possible to reduce the loss of visual
function associated with an ocular disorder, inparticular while the
ocular disorder is being treated, by reducing the loss of
photoreceptor viabilabilty and/or retinal pigment epithelial cell
viability
[0012] In one aspect, the invention provides a method preserving
the visual function of an eye of a subject with an ocular
condition, wherein a symptom of the ocular condition is the loss of
photoreceptor cell viability in the retina of the eye with the
condition. The method comprises (a) administering to the eye of the
subject an effective amount of a necrostatin and an effective
amount of an apoptosis inhibitor thereby preserving the viability
of the photoreceptor cells disposed within the retina of the eye,
and (b) then measuring the visual function (e.g., visual acuity) of
the eye after the administration of the necrosis inhibitor and the
apoptosis inhibitor. After administration of the necrosis inhibitor
and the apoptosis inhibitor the visual function (e.g., visual
acuity) of the eye may be preserved or improved relative to the
visual function of the eye prior to administration of the necrosis
inhibitor and the apoptosis inhibitor.
[0013] The ocular condition may be a condition selected from the
group consisting of AMD, RP, macular edema, diabetic retinopathy,
central areolar choroidal dystrophy, BEST disease, adult
vitelliform disease, pattern dystrophy, myopic degeneration,
central serous retinopathy, Stargardt's disease, Cone-Rod
dystrophy, North Carolina dystrophy, infectious retinitis,
inflammatory retinitis, uveitis, toxic retinitis and light-induced
toxicity. AMD may be the neovascular or the dry form of AMD.
Retinal detachment may be a rhegmatogenous, a serous, or a
tractional retinal detachment.
[0014] In another aspect, the invention provides a method of
preserving the viability of retinal pigment epithelial (RPE) cells
within the retina of a subject with an ocular condition, wherein a
symptom of the ocular condition is the loss of retinal pigment
epithelial cells in the retina of the eye with the condition. The
method comprises administering to the eye of the subject an
effective amount of a necrosis inhibitor and an apoptosis inhibitor
thereby preserving the viability of the retinal pigment epithelial
cells. The ocular condition may be selected from the group
consisting of AMD, BEST disease, myopic degeneration, Stargardt's
disease, uveitis, adult foveomacular dystrophy, fundus
falvimaculatus, multiple evanescent white dot syndrome, serpiginous
choroidopathy, acute multifocal posterior placoid epitheliopathy
(AMPPE), and other uveitis disorders.
[0015] In another aspect, the invention provides a method of
preserving the viability of photoreceptor cells disposed within a
retina of a subject with an ocular condition selected from the
group consisting of AMD, RP, macular edema, diabetic retinopathy,
central areolar choroidal dystrophy, BEST disease, adult
vitelliform disease, pattern dystrophy, myopic degeneration,
central serous retinopathy, Stargardt's disease, Cone-Rod
dystrophy, North Carolina dystrophy, infectious retinitis,
inflammatory retinitis, uveitis, toxic retinitis and light-induced
toxicity. The method comprises administering to the eye an
effective amount of a necrosis inhibitor and an effective amount of
an apoptosis inhibitor thereby to preserve the viability of the
photoreceptor cells disposed within the retina of the subject with
a condition.
[0016] In another aspect, the invention provides a method of
preserving the viability of photoreceptor cells disposed within a
retina of a mammalian eye following retinal detachment. The method
comprises administering a necrostatin and an apoptosis inhibitor to
the eye in which a region of the retina has been detached in
amounts sufficient to preserve the viability of photoreceptor cells
disposed within the region of the detached retina. When
necrostatin-1 is the only necrostatin administered, the region is
exposed to a final concentration of necrostatin-1 in the eye
greater than about 100 .mu.M.
[0017] The prevention of photoreceptor death following retinal
detachment by co-administration of an apoptotic inhibitor and a
necrostatin, e.g., necrostatin-1, at concentrations that exceed 100
.mu.M was surprising because it had been believed that
concentrations of necrostatin-1 exceeding 100 .mu.M were toxic and,
therefore, could not be administered at such a dosage. Moreover, it
was an unexpected finding that the combination of a necrostatin,
e.g., necrostatin-1, and a pan-caspase inhibitor, e.g., Z-VAD,
achieved a synergistic effect in reducing photoreceptor cell death
following retinal detachment compared to either drug alone.
[0018] In certain embodiments, the retinal detachment may be a
rhegmatogenous retinal detachment, tractional retinal detachment,
or serous retinal detachment. In other embodiments, the retinal
detachment may occur as a result of a retinal tear, retinoblastoma,
melanoma or other cancers, diabetic retinopathy, uveitis, choroidal
neovascularization, retinal ischemia, pathologic myopia, or
trauma.
[0019] In another aspect, the invention provides a method of
preserving visual function of an eye of a subject with an ocular
condition selected from the group consisting of AMD, RP, macular
edema, central areolar choroidal dystrophy, retinal detachment,
diabetic retinopathy, BEST disease, adult vitelliform disease,
pattern dystrophy, myopic degeneration, central serous retinopathy,
Stargardt's disease, Cone-Rod dystrophy, North Carolina dystrophy,
infectious retinitis, inflammatory retinitis, uveitis, toxic
retinitis and light-induced toxicity, wherein a symptom of the
ocular condition is the loss of photoreceptor cells viability in
the retina of the eye. The method comprises reducing the production
and/or activity of a RIP-1 kinase and/or RIP-3 kinase in the eye
thereby preserving the viability of the photoreceptor cells
disposed with the retina of the eye. In certain embodiments, the
reduction in the production or activity of the RIP-1 kinase and/or
the RIP-3 kinase can achieved by administering an effective amount
of RIP kinase (RIPK) inhibitor, e.g., a necrostatin.
[0020] In another aspect, the invention provides a method of
preserving the visual function of an eye of a subject with an
ocular condition, wherein a symptom of the ocular condition is the
loss of photoreceptor cell viability and/or RPE viability in the
retina of the eye. The method comprises (a) reducing the production
or activity of a RIP-1 kinase and/or a RIP-3 kinase in the eye
thereby to preserve the viability of the photoreceptor cells and/or
RPE cells disposed within the retina of the eye; and (b), after
treatment, measuring visual function (e.g., visual acuity) of the
eye. In certain embodiments, the reduction in the production or
activity of the RIP-1 kinase and/or the RIP-3 kinase can be
achieved by administering an effective amount of RIPK inhibitor,
e.g., a necrostatin. After administration of the RIPK inhibitor the
visual function of the eye may be preserved or improved relative to
the visual function of the eye prior to administration of the RIPK
inhibitor.
[0021] In another aspect, the invention provides a combination of a
necrosis inhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin)
and an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g.,
Z-VAD or IDN-6556), for use in preserving visual function of an eye
of a subject with certain ocular conditions described herein,
wherein a symptom of the ocular condition is the loss of
photoreceptor cell and/or RPE cell viability in the retina of the
eye with the condition.
[0022] In another aspect, the invention provides a combination of a
necrosis inhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin)
and an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g.,
Z-VAD or IDN-6556), for use in preserving the viability of
photoreceptor cells disposed in the retina of an eye with an ocular
condition, wherein a symptom of the ocular condition is the loss of
photoreceptor cell viability in the retina of the eye with the
condition. The ocular condition may be selected from AMD, RP,
macular edema, central areolar choroidal dystrophy, retinal
detachment, diabetic retinopathy, BEST disease, adult vitelliform
disease, pattern dystrophy, myopic degeneration, central serous
retinopathy, Stargardt's disease, Cone-Rod dystrophy, North
Carolina dystrophy, infectious retinitis, inflammatory retinitis,
uveitis, toxic retinitis and light-induced toxicity.
[0023] In another aspect, the invention provides a combination of a
necrosis inhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin)
and an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g.,
Z-VAD or IDN-6556), for use in preserving the viability of
photoreceptor cells disposed in the retina of an eye following
retinal detachment, provided that when necrostatin-1 is the only
necrostatin administered, the region of the retina that has been
detached is exposed to a final concentration of necrostatin-1 in
the eye greater than 100 .mu.M.
[0024] In another aspect, the invention provides a combination of a
necrosis inhibitor (e.g., a RIPK inhibitor, e.g., a necrostatin)
and an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g.,
Z-VAD or IDN-6556), for use in preserving the viability of retinal
pigment epithelial cells disposed in the retina of an eye with an
ocular condition, wherein a symptom of the ocular condition is the
loss of retinal pigment epithelial cell viability in the retina of
the eye with the condition. The ocular condition may be selected
from the group consisting of AMD, BEST disease, myopic
degeneration, Stargardt's disease, uveitis, adult foveomacular
dystrophy, fundus falvimaculatus, multiple evanescent white dot
syndrome, serpiginous choroidopathy, acute multifocal posterior
placoid epitheliopathy (AMPPE), and other uveitis disorders.
[0025] In each of the foregoing methods, the necrosis inhibitor can
be a RIP kinase inhibitor, for example, a necrostatin. In certain
embodiments of the foregoing methods, the necrostatin is
necrostatin-1, necrostatin-2, necrostatin-3, necrostatin-4,
necreostatin-5, necrostatin-7, or a combination thereof.
[0026] In certain embodiments when a necrostatin is administered,
the necrostatin is administered to provide a final concentration of
necrostatin in the eye greater than about 100 .mu.M. For example,
the final concentration of necrostatin in the eye may range from
about 150 .mu.M to about 1000 .mu.M, from about 200 .mu.M to about
800 .mu.M or from about 200 .mu.M to about 600 .mu.M. In certain
embodiments, the final concentration of necrostatin in the eye is
about 400 .mu.M. In other embodiments when a necrostatin is
administered, from about 0.05 mg to about 2 mg, 0.1 mg to about 1
mg, from about 0.2 mg to about 1 mg, or from about 0.2 mg to about
0.8 mg, of necrostatin can be administered locally to the eye of a
mammal. In an exemplary embodiment, about 0.5 mg of necrostatin can
be administered locally to the eye of a mammal.
[0027] In certain embodiments when a a pan-caspase inhibitor is
administered, the pan-caspase inhibitor is administered to provide
a final concentration of the pan-caspase inhibitor in eye greater
than about 100 .mu.M. For example, the final concentration of
pan-caspase inhibitor in the eye may range from about 150 .mu.M to
about 500 .mu.M or from about 200 .mu.M to about 400 .mu.M. In
certain embodiments, the final concentration of the pan-caspase
inhibitor in the eye is about 300 .mu.M. Exemplary pan-caspase
inhibitors include zVAD, IDN-6556 or a combination thereof. In
other embodiments, from about 0.05 mg to about 1.5 mg, from about
0.15 mg to about 1.5 mg, from about 0.2 mg to about 1 mg, from
about 0.2 mg to about 0.8 mg, from about 0.4 mg to about 1 mg, or
from about 0.5 mg to about 0.8 mg, of the pan-caspase inhibitor can
be administered locally to the eye of a mammal. In an exemplary
embodiment, about 0.7 mg of a pan-caspase inhibitor can be
administered locally to the eye of a mammal.
[0028] The necrosis inhibitor, e.g., a necrostatin, and/or the
apoptosis inhibitor may be administered to the eye by intraocular,
intravitreal, subretinal or trasscleral administration. The
necrosis inhibitor, e.g., a necrostatin, and/or the apoptosis
inhibitor may be solubilized in a viscoelastic carrier that is
introduced into the eye. In other embodiments, the necrosis
inhibitor, e.g., a necrostatin, and/or the apoptosis inhibitor may
be administered systemically.
[0029] It is understood that the necrosis inhibitor, e.g., a
necrostatin, and/or the apoptosis inhibitor may be administered
sequentially or simultaneously. The necrosis inhibitor, e.g., a
necrostatin, and the apoptosis inhibitor may be administered in the
same or different carriers.
[0030] In certain embodiments, when the ocular condition is a
retinal detachment, the necrostatin and/or the apoptosis inhibitor
may be administered to the subject prior to reattachment and/or
after reattachment of the retina or a region of the retina that has
become detached.
[0031] In each of the foregoing methods and compositions, the
necrostatin can be selected from one or more of the following
necrostatins. For example, in certain embodiments, the necrostatin
is a Nec-1 related compound of Formula I:
##STR00001##
[0032] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein
[0033] X is O or S;
[0034] R.sub.1 is hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxyl, or halogen; and
[0035] R.sub.2 is hydrogen or C.sub.1-C.sub.6alkyl.
[0036] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-A:
##STR00002##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
or optical isomers or racemic mixtures thereof, wherein R.sub.1 is
H, alkyl, alkoxyl, or a halogen and R.sub.2 is H or an alkyl.
[0037] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-B:
##STR00003##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0038] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-C:
##STR00004##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0039] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-D:
##STR00005##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0040] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-E:
##STR00006##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein R.sub.1 is H, alkyl, alkoxyl, or a halogen (for example, F,
Cl, Br or I) and R.sub.2 is H or an alkyl.
[0041] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-F:
##STR00007##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0042] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-1 related compound of Formula I-G:
##STR00008##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0043] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-2 related compound of Formula II:
##STR00009##
[0044] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein:
[0045] X is --CH.sub.2--, --C(H)(R.sub.14)--, --C(.dbd.S)--,
--C(.dbd.NH)--, or --C(O)--;
[0046] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, R.sub.9, and R.sub.10 each represent
independently hydrogen, acyl, acetyl, alkyl, halogen, amino,
C.sub.1-C.sub.6alkoxyl, nitro, --C(O)R.sub.12, --C(S)R.sub.12,
--C(O)OR.sub.12, --C(O)NR.sub.12R.sub.13, --C(S)NR.sub.12R.sub.13,
or --S(O.sub.2)R.sub.12;
[0047] R.sub.11 is hydrogen, acyl, acetyl, alkyl, or acylamino;
[0048] R.sub.12 and R.sub.13 each represent independently hydrogen,
an optionally substituted alkyl, an optionally substituted aryl, an
optionally substituted heteroaryl, an optionally substituted
aralkyl, or an optionally substituted heteroaralkyl;
[0049] R.sub.14 is acyl, acetyl, alkyl, halogen, amino, acylamino,
nitro, --SR.sub.11, --N(R.sub.11).sub.2, or --OR.sub.11;
[0050] the bond indicated by (a) can be a single or double bond;
and
[0051] the bond indicated by (b) can be a single or double
bond.
[0052] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-2 related compound of Formula IIA:
##STR00010##
[0053] or a pharmaceutically acceptable salt thereof, wherein:
[0054] R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sub.7, and R.sub.10
each represent independently hydrogen, alkyl, halogen, amino, or
methoxyl; and
[0055] R.sub.3, R.sub.4, R.sub.8, and R.sub.9 are
C.sub.1-C.sub.6alkoxyl.
[0056] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-3 related compound of Formula III:
##STR00011##
[0057] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein:
[0058] Z is --CH.sub.2--, --CH.sub.2CH.sub.2--, --O--, --S--,
--S(O)--, --S(O.sub.2)--, or --N(R.sub.7)--;
[0059] R.sub.1, R.sub.3, and R.sub.5 each represent independently
for each occurrence hydrogen, halogen, hydroxyl, amino,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxy-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfinyl,
C.sub.1-C.sub.6alkylsulfinyl-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonyl-C.sub.1-C.sub.6alkyl, aryl, aralkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl;
[0060] R.sub.2 and R.sub.4 are C.sub.1-C.sub.6alkoxy;
[0061] R.sub.6 is --C(O)R.sub.8, --C(S)R.sub.8, --C(O)OR.sub.8,
--C(O)NR.sub.8R.sub.9, --C(S)NR.sub.8R.sub.9, --C(NH)R.sub.8, or
--S(O.sub.2)R.sub.8;
[0062] R.sub.7 is alkyl, aralkyl, or heteroaralkyl;
[0063] R.sub.8 and R.sub.9 each represent independently hydrogen,
C.sub.1-C.sub.6alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl; and n represents independently for each occurrence
0, 1, or 2.
[0064] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-4 related compound of Formula IV:
##STR00012##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0065] R.sub.1 is
##STR00013##
[0066] R.sub.2 and R.sub.3 each represent independently for each
occurrence hydrogen or methyl;
[0067] R.sub.4 represents independently for each occurrence
halogen, hydrogen, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl, or
C.sub.2-C.sub.4alkynyl;
[0068] R.sub.5 is C.sub.1-C.sub.4alkyl;
[0069] R.sub.6 is hydrogen, halogen, or --CN;
[0070] R.sub.7 is hydrogen or C.sub.1-C.sub.4alkyl;
[0071] R.sub.8 is C.sub.1-C.sub.6alkyl, or R.sub.8 taken together
with R.sub.9, when present, forms a carbocyclic ring;
[0072] R.sub.9 is hydrogen or C.sub.1-C.sub.6alkyl, or R.sub.9
taken together with R.sub.8 forms a carbocyclic ring;
[0073] R.sub.10 is hydrogen or C.sub.1-C.sub.6alkyl;
[0074] A is phenylene or a 5-6 membered heteroarylene;
[0075] X is N or --C(R.sub.9)--;
[0076] Y is N or --C(R.sub.10)--;
[0077] Z is S or O; and
[0078] m and n each represent independently 1, 2, or 3.
[0079] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-4 related compound of Formula IV-A:
##STR00014##
or a pharmaceutically acceptable salt thereof.
[0080] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-5 related compound of Formula V:
##STR00015##
[0081] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein:
[0082] A is a saturated or unsaturated 5-6 membered carbocyclic
ring;
[0083] X is a bond or C.sub.1-C.sub.4alkylene;
[0084] R.sub.1 is C.sub.1-C.sub.6 alkyl, halogen, hydroxyl,
C.sub.1-C.sub.6alkoxyl, --N(R.sub.4).sub.2, --C(O)R.sub.4,
CO.sub.2R.sub.4, or C(O)N(R.sub.4).sub.2;
[0085] R.sub.2 is
##STR00016##
[0086] R.sub.3 is --C.sub.1-C.sub.6alkylene-CN, --CN,
C.sub.1-C.sub.6alkyl, or C.sub.2-C.sub.6alkenyl;
[0087] R.sub.4 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl;
[0088] R.sub.5 represents independently for each occurrence
C.sub.1-C.sub.6alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2;
[0089] B is a 5-6 membered heterocyclic or carbocylic ring; and
[0090] n and p each represent independently 0, 1, or 2.
[0091] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-5 related compound of Formula V-A:
##STR00017##
[0092] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein:
[0093] R.sub.1 is C.sub.1-C.sub.6alkyl, halogen, hydroxyl,
C.sub.1-C.sub.6alkoxyl, or --N(R.sub.4).sub.2;
[0094] R.sub.2 is
##STR00018##
[0095] R.sub.3 is --C.sub.1-C.sub.6alkylene-CN;
[0096] R.sub.4 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl;
[0097] R.sub.5 represents independently for each occurrence
C.sub.1-C.sub.6alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2;
[0098] B is a 5-6 membered heterocyclic or carbocylic ring; and
[0099] n and p each represent independently 0, 1, or 2.
[0100] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-7 related compound of Formula VII:
##STR00019##
[0101] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein:
[0102] R.sub.1, R.sub.2, and R.sub.3 each represent independently
hydrogen or C.sub.1-C.sub.4alkyl;
[0103] R.sub.4 is
##STR00020##
[0104] R.sub.4 is R or
[0105] R.sub.5 and R.sub.6 each represent independently for each
occurrence halogen, C.sub.1-C.sub.6alkyl, hydroxyl,
C.sub.1-C.sub.6alkoxyl, --N(R.sub.7).sub.2, --NO.sub.2,
--S--C.sub.1-C.sub.6alkyl, --S-aryl,
--SO.sub.2--C.sub.1-C.sub.6alkyl, --SO.sub.2-aryl, --C(O)R.sub.7,
--CO.sub.2R.sub.7, --C(O)N(R.sub.7).sub.2, heterocycloalkyl, aryl,
or heteroaryl;
[0106] R.sub.7 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl; or two
occurrences of R.sub.7 attached to the same nitrogen atom are taken
together with the nitrogen atom to which they are attached to form
a 3-7 membered heterocyclic ring;
[0107] A is a 5-6 membered heterocyclic ring; and
[0108] p is 0, 1, or 2.
[0109] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-7 related compound of Formula VIII:
##STR00021##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0110] each X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 is selected, independently, from N or CR.sup.X1;
[0111] each Y.sup.1, Y.sup.2, and Y.sup.3 is selected,
independently, from O, S, NR.sup.Y1, or CR.sup.Y2R.sup.Y3;
[0112] each Z.sup.1 and Z.sup.2 is selected, independently, from O,
S, or NR.sup.Z;
[0113] each R.sup.Y1 and R.sup.Z1 is selected, independently, from
H, optionally substituted C.sub.1-6alkyl, optionally substituted
C.sub.2-6alkenyl, optionally substituted C.sub.2-6alkynyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, --C(.dbd.O)R.sup.5A, --C(.dbd.O)OR.sup.5A, or
--C(.dbd.O)NR.sup.5AR.sup.6A;
[0114] each R.sup.X1, R.sup.Y2, and R.sup.Y3 is selected,
independently, from H, halogen, CN, NC, NO.sub.2, N.sub.3,
OR.sup.3, SR.sup.3, NR.sup.3R.sup.4, --C(.dbd.O)R.sup.5A,
--C(.dbd.O)OR.sup.5A, --C(.dbd.O)NR.sup.5AR.sup.6A,
--S(.dbd.O)R.sup.5A, --S(.dbd.O).sub.2R.sup.5A,
--S(.dbd.O).sub.2OR.sup.5A, --S(.dbd.O).sub.2NR.sup.5AR.sup.6A,
optionally substituted C.sub.1-6alkyl, optionally substituted
C.sub.2-6alkenyl, optionally substituted C.sub.2-6alkynyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, or optionally
substituted heteroaryl;
[0115] each R.sup.1, R.sup.2 R.sup.5A, R.sup.5B, R.sup.6A, and
R.sup.6B is selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6alkenyl, optionally
substituted C.sub.2-6alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
or optionally substituted heteroaryl; or R.sup.5A and R.sup.6A, or
R.sup.5B and R.sup.6B combine to form a heterocyclyl; and
[0116] each R.sup.3 and R.sup.4 is selected from H, optionally
substituted C.sub.1-6 alkyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, --C(.dbd.O)R.sup.5B,
--C(.dbd.S)R.sup.5B, --C(.dbd.NR.sup.6B)R.sup.5B,
--C(.dbd.O)OR.sup.5B, --C(.dbd.O)NR.sup.5BR.sup.6B,
--S(.dbd.O)R.sup.5B, --S(.dbd.O).sub.2R.sup.5B,
--S(.dbd.O).sub.2OR.sup.5B, or --S(.dbd.O).sub.2NR.sup.5BR.sup.6B.
In certain embodiments when R.sub.1 is H, X.sup.1, X.sup.2, and
X.sup.4 are each CH, X.sup.3, X.sup.5, and X.sup.6 are each N,
Y.sup.1 and Y.sup.3 are each S, Y.sup.2 is NH, Z.sup.1 is NH, and
Z.sup.2 is O, then R.sup.2 is not 4-fluorophenyl.
[0117] In each of the foregoing methods and compositions, the
necrostatin can be a Nec-4 related compound of Formula IX:
##STR00022##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0118] X.sub.1 and X.sub.2 are, independently, N or CR.sup.4;
[0119] X.sub.3 is selected from O, S, NR.sup.5, or
--(CR.sup.5).sub.2;
[0120] Y is selected from C(O) or CH.sub.2; and
[0121] Z is (CR.sup.6R.sup.7).sub.n;
[0122] R.sup.1 is selected from H, halogen, optionally substituted
C.sub.1-6alkyl, or optionally substituted C.sub.1-6cycloalkyl, or
optionally substituted aryl;
[0123] R.sup.1 is selected from H or optionally substituted
C.sub.1-6alkyl;
[0124] R.sup.3 is optionally substituted aryl;
[0125] each R.sup.4 is selected from H, halogen, carboxamido,
nitro, cyano, optionally substituted C.sub.1-6alkyl, or optionally
substituted aryl;
[0126] R.sup.5 is selected from H, halogen, optionally substituted
C.sub.1-6alkyl, or optionally substituted aryl;
[0127] each R.sup.6 and R.sup.7 is, independently, selected from H,
optionally substituted C.sub.1-6alkyl, or aryl; and
[0128] n is 0, 1, 2, or 3. In certain embodiments, when X.sub.1 and
X.sub.2 are N, X.sub.3 is S, Y is C(O), Z is CH.sub.2, R.sup.2 is
H, and R.sup.3 is 2-chloro-6-fluoro-phenyl, then R.sup.1 is not
methyl.
[0129] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0130] The objects and features of the invention may be more fully
understood by reference to the drawings described herein.
[0131] FIG. 1A provides a schematic representation of the RIP-1
signaling pathway. FIG. 1B-D depict graphs and photographs that
show the increase in RIP-3 (FIG. 1B) and RIP-1 (FIG. 1C) expression
after retinal detachment, as determined by quantitative real-time
PCR analysis, and by Western blot analysis (FIG. 1D).
[0132] FIG. 2 depicts photographs showing results of in situ
hybridization analysis of RIP-3.
[0133] FIG. 3A-B are photographs of immunoblots of an
immunoprecipitation of RIP-1 from retinal lysates (FIG. 3A) and
phosphorylation of RIP-1 after retinal detachment (FIG. 3B).
[0134] FIG. 4A-C are photographs and graphs showing that
necrostatin-1 (Nec-1) combined with Z-VAD prevents photoreceptor
loss 3 and 5 days after retinal detachment.
[0135] FIG. 5A-B are graphs showing that intravitreal injection of
Z-VAD+Nec-1 at one day after retinal detachment prevents
photoreceptor loss when measured three days after retinal
detachment.
[0136] FIG. 6A-F are graphs showing quantification of
TUNEL-positive photoreceptors (FIGS. 6A, 6C and 6E) and ONL
thickness ratio (FIGS. 6B, 6D and 6F) on day three after retinal
detachment.
[0137] FIG. 7A-D are transmission electron microscope (TEM)
photomicrographs and FIG. 7E is a graph depicting the involvement
of programmed necrosis during retinal detachment induced
photoreceptor death, wherein cells denoted as A represent apoptotic
cells, and cells denoted as N represent necrotic cells.
[0138] FIG. 8A-D are photographs of PI staining (FIGS. 8A and 8C)
and graphs showing quantification of PI-positive photoreceptors
(FIGS. 8B and 8D) on day three after retinal detachment in retina
treated with vehicle, Z-VAD or Z-VAD+Nec-1 in wild-type (FIGS. 8A
and 8B) and RIP-3-/- retinas (FIGS. 8C and 8D).
[0139] FIG. 9A-C are TUNEL photographs (FIG. 9A) and graphs (FIGS.
9B and 9C) showing the reduction of photoreceptor cell death
following retinal detachment in RIP-3-/- mice and in RIP-3-/- mice
treated with Z-VAD.
[0140] FIG. 10A-B are TEM photographs in the ONL on day 3 after
retinal detachment in wild-type and RIP-3-/- retina treated with
vehicle, Z-VAD, Nec-1, or Z-VAD+Nec-1.
[0141] FIG. 11A-F are photographs and graphs showing that Nec-1
suppressed the inflammatory response after retinal detachment.
[0142] FIG. 12A-C are graphs showing quantitative real-time PCR
analysis for MCP-1 (FIG. 12A), TNF-.alpha. (FIG. 12B), and Fas-L
(FIG. 12C) in control retina without retinal detachment (n=9) and
in retina three days after retinal detachment with treatment of
vehicle (n=9), Z-VAD (n=8) or Z-VAD+Nec-1 (n=9). *, P<0.05; **,
P<0.01. FIG. 13A-B are graphs showing results from an ELISA for
MCP-1 (FIG. 13A) and TNF-.alpha. (FIG. 13B) in retina without
retinal detachment (n=5) and in retina three days after retinal
detachment with treatment of vehicle (n=5), Z-VAD (n=5) or
Z-VAD+Nec-1 (n=6).
[0143] FIG. 14 A-F are photographs and graphs showing RIP kinase
inhibition prevents ROS production and AIF nuclear translocation
after retinal detachment.
[0144] FIG. 15A-C are schematic representations showing a proposed
mechanism of photoreceptor loss after retinal detachment. (FIG.
15A) After retinal detachment, photoreceptor death is caused mainly
by apoptosis. (FIG. 15B) Caspase inhibition by Z-VAD decreases
apoptosis but promotes RIP-mediated programmed necrosis. (FIG. 15C)
Blockade of both caspases and RIP kinases is essential for
effective prevention of photoreceptor loss.
[0145] FIG. 16 is a photograph of an immunoblot from a Western blot
analysis for phospho-NF.kappa.B p65 in control retina without a
retinal detachment and in retina three days after retinal
detachment with treatment of vehicle, Z-VAD or Z-VAD+Nec-1
(lane-loading differences were normalized by the level of
.beta.-tubulin).
[0146] FIG. 17A-B are photographs of immunoblots from a (FIG. 17A)
Western blot analysis for light chain 3 in control retina without
retinal detachment and in retina three days after retinal
detachment with treatment of vehicle or Z-VAD+Nec and a (FIG. 17B)
Western blot analysis of phosphorylated ribosomal protein S6 in
control retina without retinal detachment and in retina 3 days
after retinal detachment.
[0147] FIG. 18A is a graph showing quantitative real-time PCR
analysis for TNF-.alpha. in control retina without retinal
detachment (n=9) and in retina three days after retinal detachment
with treatment of vehicle (n=9), Z-VAD (n=8) or Z-VAD+Nec-1 (n=9).
*, P<0.05; **, P<0.01.
[0148] FIG. 18B is a graph showing results from an ELISA for
TNF-.alpha. in retina without retinal detachment (n=5) and in
retina three days after retinal detachment with treatment of
vehicle (n=5), Z-VAD (n=5) or Z-VAD+Nec-1 (n=6). FIG. 18C-D are a
photograph and a graph showing DAPI staining (FIG. 18C) and
quantification of the outer nuclear layer (ONL) thickness ratio
(FIG. 18D) in detached retina treated with anti-TNF-.alpha.
antibody or control antibody on day three after retinal detachment
(n=4 each). *, P<0.05.
[0149] FIG. 19 is another schematic diagram of the RIP-1 signaling
pathway.
[0150] FIG. 20A is a schematic representation of the TLR3 and RIP
signaling pathway. FIG. 20B is a photograph showing significant
loss of photoreceptors and RPE in the ONL following poly I:C
treatment.
[0151] FIG. 21A-D are graphs showing RIP-1 (FIG. 21A) and RIP-3
(FIG. 21B) expression in the retina, as well as RIP-3 expression in
the RPE cells (FIG. 21C) and macrophages (FIG. 21D), following poly
I:C treatment, as determined by quantitative real-time PCR
analysis.
[0152] FIG. 22A-D are photographs and graphs showing quantification
of TUNEL-positive photoreceptors (FIG. 22A and FIG. 22B) and ONL
thickness (FIG. 22C and FIG. 22D) in RIP-3-/- mice two days after
treatment with poly I:C.
[0153] FIG. 23A-B are photographs of PI staining and graph showing
quantification of PI-positive photoreceptors in RIP-3-/- mice two
days after treatment with poly I:C.
[0154] FIG. 24A-B are graphs showing quantification of
TUNEL-positive photoreceptors (FIG. 24A) and ONL thickness (FIG.
24B) in mice treated with poly I:C.
[0155] FIG. 25A are photographs showing the development of
drusen-like lesions, retinal degeneration and choroidal
neovascularization in CX3CR1-/-CCL2-/- double knockout mice. FIG.
25B provides graphs showing RIP-1 and RIP-3 expression in the
retina of CX3CR1-/-CCL2-/- double knockout mice at 5 weeks (5 w)
and 5 months (5M) of age.
[0156] FIG. 26 provides photographs showing the development of
drusen-like lesions in CX3CR1-/-CCL2-/- RIP-3+/+ knockout mice
(first row), CX3CR1-/-CCL2-/- RIP-3+/- knockout mice (second row),
and CX3CR1-/-CCL2-/- RIP-3-/- triple knockout mice (third row).
[0157] FIG. 27 provides graphs showing expression of various
inflammatory cytokines in the retina of WT mice, CX3CR1-/-CCL2-/-
double knockout mice, and CX3CR1-/-CCL2-/- RIP-3-/- triple knockout
mice at 2 months of age.
[0158] FIG. 28 provides photographs showing loss of photoreceptors
and RPE in the ONL in rd10 mice at postnatal day 28.
[0159] FIG. 29A-B are graphs showing RIP-1 and RIP-3 expression in
the retina of rd10 mice at postnatal day 21, 28, and 35.
[0160] FIG. 30A-B are photographs and a graph showing ONL thickness
in rd10 mice following Z-VAD+NEC-1 daily intraperitoneal injections
from postnatal day 25 to postnatal day 28.
[0161] FIG. 31A-B are photographs and a graph showing ONL thickness
in rd10 RIP-3 double mutant mice.
[0162] FIG. 32A-B are graphs showing RIP-1 (FIG. 32A) and RIP-3
(FIG. 32B) expression in poly I:C treated RPE cells.
[0163] FIG. 33A is a graph showing the viability of RPE cells
treated with poly I:C in combination with Z-VAD or Z-VAD+Nec1. FIG.
33B is a graph showing the viability of WT or RIP-3-/- RPE cells
treated with poly I:C in combination with Z-VAD.
DETAILED DESCRIPTION
[0164] The invention relates to methods and composition for
preserving the viability of photoreceptor cells and/or retinal
pigment epithelial cells disposed within a retina of an eye of a
subject with certain ocular conditions, wherein the viability of
the photoreceptor cells and/or the retinal pigment epithelial cells
are affected by the ocular disorder. As it result, using the
methods and compositions described herein, it may be possible to
preserve or improve visual function in the eye by maintaining
photoreceptor viability and/or retinal pigment epithelial cell
viability while the underlying ocular condition is being
treated.
[0165] As demonstrated herein, programmed necrosis appears to be a
critical mechanism of photoreceptor death in certain ocular
disorders, for example, AMD, RP, and following retinal detachment
in the presence of an apoptosis inhibitor, e.g., a pan-caspase
inhibitor. As depicted in FIG. 1A, there are two pathways for cell
death--apoptosis and necrosis, which appear to be mediated by
RIP-1, a serine/threonine kinase. RIP-1 can act on or modulate
NF-.kappa.B to affect cell survival. RIP-1 can also form a complex
with the Fas-associated death domain (FADD) and caspase-8 in
response to death receptor stimulation to modulate the apoptotic
pathway of cell death. In addition, RIP-1 can form a complex with
RIP-3 to modulate the necrotic pathway of cell death. As shown in
FIG. 1A, modulation of the apoptotic pathway (e.g., with a
pan-caspase inhibitor, e.g., Z-VAD) can also affect the necrotic
pathway.
[0166] The methods and compositions described herein are directed
to therapies that target both the necrotic and apoptotic pathways
of programmed cell death. In particular, the methods and
compositions disclosed herein facilitate a combination therapy
where a necrosis inhibitor, e.g, a necrostatin (e.g., necrostatin-1
or necrostatin-4), can be administered either alone or in
combination (either sequentially or simultaneously) with an
apoptosis inhibitor e.g., a pan-caspase inhibitor (e.g., Z-VAD or
IDN-6556). In certain embodiments, the disclosed methods
surprisingly use necrostatins at concentrations higher than those
previously thought to be clinically tolerable. Moreover, it has
been demonstrated that the combination of a necrostatin, e.g.,
necrostatin-1 or necrostatin-4, and a pan-caspase inhibitor, e.g.,
Z-VAD or IDN-6556, produce a superior effect, e.g., a synergistic
effect in reducing photoreceptor cell death in AMD, RP and
following retinal detachment, compared to either drug alone.
[0167] For convenience, certain terms in the specification,
examples, and appended claims are collected in this section.
[0168] As used herein, the term "cell death" is understood to mean
the death of a cell by either apoptosis or necrosis.
[0169] As used herein, the term "apoptosis" is understood to mean
caspase-dependent cell death, which is characterized by any of the
following properties: cell shrinkage, nuclear condensation, DNA
fragmentation or membrane blebbing.
[0170] As used herein, the term "apoptosis inhibitor" is understood
to mean any agent that, when administered to a mammal, reduces
apoptotic cell death in photoreceptor and/or RPE cells. For
example, it is understood that certain useful apoptosis inhibitors
act by reducing or eliminating the activity of one or more members
of the intrinsic or extrinsic or common apoptotic pathways.
Furthermore, it is understood that an agent that either directly or
indirectly affects the activity of one or more caspases (e.g., a
pan-caspase inhibitor) is considered to be an apoptosis inhibitor.
It is understood that a caspase inhibitor can affect the activity
of a caspase either directly by modulating a specific caspase in
the apoptotic pathway or indirectly by modulating a downstream
caspase present in the apoptotic pathway.
[0171] As used herein, the term "pan-caspase inhibitor" is
understood to mean a broad-spectrum caspase inhibitor that inhibits
at least two, preferably at least three different caspases (e.g.,
caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6,
caspase-7, caspase-8, caspase-9, caspase-10, caspase-11,
caspase-12, caspase-13, and/or caspase-14. Z-VAD (also known as
Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and
carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone)
is an exemplary pan-caspase inhibitor and is available from R&D
Systems (Cat. No. FMK001) and Promega (Cat. No. G7231). Other
exemplary pan-caspase inhibitors that may be used include IDN-6556
(also known as "PF-3,491,390") available from Conatus
Pharmaceuticals, Inc. (formerly Idun Pharmaceuticals, Inc.), VX-799
available from Vertex Pharmaceuticals, Inc., MX1013 available Maxim
Pharmaceuticals, Inc., Xyz033mp available from LG Chemical, Inc.,
all of which are described, for example, in Linton, S. D. (2005)
CURRENT TOPICS IN MEDICAL CHEM. 5:1697-1717. It is understood that
a "pan-caspase inhibitor" may also be a cocktail (e.g., a
combination) of caspase inhibitors including two or more of
specific caspase inhibitors (e.g., synthetic or endogenous caspase
inhibitors).
[0172] As used herein, the term "necrosis" is understood to mean
caspase-independent cell death characterized by any of the
following properties: cellular and/or organelle swelling, plasma
membrane rupture, or discontinuity in plasma, nuclear and/or
organelle membranes. As used herein, the terms "necroptosis" and
"programmed necrosis" refer to a form of necrosis and is understood
to mean one form of programmed or regulated necrosis, and in
certain embodiments, necroptosis is mediated by the
serine/threonine kinase activity of receptor interacting protein
(RIP) kinases, for example, RIP-1 kinase and/or RIP-3 kinase.
[0173] As used herein, the term "necrosis inhibitor" is understood
to mean an agent, which, when administered to a mammal, reduces
necrotic cell death in photoreceptor and/or RPE cells. For example,
it is understood that certain necrosis inhibitors act by reducing
or inhibiting necroptosis or programmed necrosis. A necrosis
inhibitor can be an agent that modulates the production and/or
activity of one or more RIP kinases (e.g., RIP-1 kinase and/or
RIP-3 kinase). For example, an inhibitor of RIP-1 kinase is
understood to modulate the activity of RIP-1 kinase as well as
downstream RIP kinases, e.g., RIP-3 kinase, in the necrosis
cascade. Accordingly, a RIP-1 kinase inhibitor is also understood
to modulate RIP-3 kinase activity.
[0174] As used herein, the term "necrostatin" or "nec" is
understood to mean an inhibitor of caspase-independent cell death
or necroptosis. Exemplary necrostatins include necrostatin-1
("Nec-1"), necrostatin-2 ("Nec-2"), necrostatin-3 ("Nec-3"),
necrostatin-4 ("Nec-4"), necrostatin-5 ("Nec-5") and necrostatin-7
("Nec-7").
[0175] In certain embodiments, the necrostatin is a Nec-1 related
compound of Formula I:
##STR00023##
[0176] or a pharmaceutically acceptable salt, ester, or prodrug
thereof, wherein
[0177] X is O or S;
[0178] R.sub.1 is hydrogen, C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkoxyl, or halogen; and
[0179] R.sub.2 is hydrogen or C.sub.1-C.sub.6alkyl.
[0180] In certain embodiments, X is O. In certain embodiments,
R.sub.1 is hydrogen or halogen (such as chlorine). In certain
embodiments, R.sub.2 is a methyl or ethyl. In certain other
embodiments, R.sub.1 is hydrogen or Cl, and R.sub.2 is a
methyl.
[0181] In certain embodiments, the necrostatin is a Nec-1 related
compound of Formula I-A, shown below:
##STR00024##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
or optical isomers or racemic mixtures thereof, wherein R.sub.1 is
H, alkyl, alkoxyl, or a halogen (for example, F, Cl, Br or I) and
R.sub.2 is H or an alkyl. In certain embodiments, R.sub.1 is H or
Cl. In certain other embodiments, R.sub.2 is a methyl or ethyl. In
certain other embodiments, R.sub.1 is H or Cl, and R.sub.2 is a
methyl.
[0182] In certain other embodiments, the necrostatin is a Nec-1
related compound of Formula I-B, shown below:
##STR00025##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0183] In certain other embodiments, the necrostatin is a Nec-1
related compound of Formula I-C, shown below:
##STR00026##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0184] In certain other embodiments, the necrostatin is a Nec-1
related compound of Formula I-D, shown below:
##STR00027##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0185] In certain other embodiments, the necrostatin is a Nec-1
related compound of Formula I-E, shown below:
##STR00028##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein R.sub.1 is H, alkyl, alkoxyl, or a halogen (for example, F,
Cl, Br or I) and R.sub.2 is H or an alkyl. In certain embodiments,
R.sub.1 is H or Cl. In certain other embodiments, R.sub.2 is a
methyl or ethyl. In certain other embodiments, R.sub.1 is H or Cl,
and R.sub.2 is a methyl.
[0186] In certain other embodiments, the necrostatin is a Nec-1
related compound of Formula I-F, shown below:
##STR00029##
or a pharmaceutically acceptable salt, ester, or prodrug thereof.
In certain other embodiments, the necrostatin is a Nec-1 related
compound of Formula I-G, shown below:
##STR00030##
or a pharmaceutically acceptable salt, ester, or prodrug
thereof.
[0187] The Nec-1 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in Degterev et al. in Nature Chemical Biology, (2005), vol. 1,
112-119; Degterev et al. in Nature Chemical Biology, (2008), vol.
4, 313-321; and International Patent Application Publication No. WO
2007/075772, all of which are hereby incorporated by reference.
[0188] In certain embodiments, the necrostatin is a Nec-2 related
compound of Formula II:
##STR00031##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0189] X is --CH.sub.2--, --C(H)(R.sub.14)--, --C(.dbd.S)--,
--C(.dbd.NH)--, or --C(O)--;
[0190] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, R.sub.9, and R.sub.10 each represent
independently hydrogen, acyl, acetyl, alkyl, halogen, amino,
C.sub.1-C.sub.6alkoxyl, nitro, --C(O)R.sub.12, --C(S)R.sub.12,
--C(O)OR.sub.12, --C(O)NR.sup.12R.sub.13, --C(S)NR.sub.12R.sub.13,
or --S(O.sub.2)R.sub.12;
[0191] R.sub.11 is hydrogen, acyl, acetyl, alkyl, or acylamino;
[0192] R.sub.12 and R.sub.13 each represent independently hydrogen,
an optionally substituted alkyl, an optionally substituted aryl, an
optionally substituted heteroaryl, an optionally substituted
aralkyl, or an optionally substituted heteroaralkyl;
[0193] R.sub.14 is acyl, acetyl, alkyl, halogen, amino, acylamino,
nitro, --SR.sub.11, --N(R.sub.11).sub.2, or --OR.sub.11;
[0194] the bond indicated by (a) can be a single or double bond;
and
[0195] the bond indicated by (b) can be a single or double
bond.
[0196] In certain embodiments, X is --C(O)--. In certain
embodiments, R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sub.7, and
R.sub.10 each represent independently hydrogen, acyl, alkyl,
halogen, or amino. In certain embodiments, R.sub.3, R.sub.4,
R.sub.8, and R.sub.9 are C.sub.1-C.sub.6alkoxyl. In certain
embodiments, the bond indicated by (a) is a double bond; and the
bond indicated by (b) is a double bond. In certain embodiments,
when each of R.sub.1, R.sub.4, R.sub.5, R.sub.6, R.sub.9 and
R.sub.10 is hydrogen and each of R.sub.2, R.sub.3, R.sub.7, and
R.sub.8 is methoxyl, then X is not --C(O)--, --CH.sub.2--, or
--CH(OH)--.
[0197] In certain other embodiments, the necrostatin is a Nec-2
related compound of Formula II-A:
##STR00032##
or a pharmaceutically acceptable salt thereof, wherein:
[0198] R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sub.7, and R.sub.10
each represent independently hydrogen, alkyl, halogen, amino, or
methoxyl; and
[0199] R.sub.3, R.sub.4, R.sub.8, and R.sub.9 are
C.sub.1-C.sub.6alkoxyl.
[0200] In certain other embodiments, the Nec-2 related compound
is
##STR00033##
or a pharmaceutically acceptable salt thereof.
[0201] The Nec-2 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in International Patent Application Publication No. WO 2007/075772,
which is hereby incorporated by reference.
[0202] In certain embodiments, the necrostatin is a Nec-3 related
compound of Formula III:
##STR00034##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0203] Z is --CH.sub.2--, --CH.sub.2CH.sub.2--, --O--, --S--,
--S(O)--, --S(O.sub.2)--, or --N(R.sub.7)--;
[0204] R.sub.1, R.sub.3, and R.sub.5 each represent independently
for each occurrence hydrogen, halogen, hydroxyl, amino,
C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy,
C.sub.1-C.sub.6alkoxy-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkanoyl, C.sub.1-C.sub.6alkylsulfinyl,
C.sub.1-C.sub.6alkylsulfinyl-C.sub.1-C.sub.6alkyl,
C.sub.1-C.sub.6alkylsulfonyl,
C.sub.1-C.sub.6alkylsulfonyl-C.sub.1-C.sub.6alkyl, aryl, aralkyl,
heterocycloalkyl, heteroaryl, or heteroaralkyl;
[0205] R.sub.2 and R.sub.4 are C.sub.1-C.sub.6alkoxy;
[0206] R.sub.6 is --C(O)R.sub.8, --C(S)R.sub.8, --C(O)OR.sub.8,
--C(O)NR.sub.8R.sub.9, --C(S)NR.sub.8R.sub.9, --C(NH)R.sub.8, or
--S(O.sub.2)R.sub.8;
[0207] R.sub.7 is alkyl, aralkyl, or heteroaralkyl;
[0208] R.sub.8 and R.sub.9 each represent independently hydrogen,
C.sub.1-C.sub.6alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl; and
[0209] n represents independently for each occurrence 0, 1, or
2.
[0210] In certain embodiments, Z is --CH.sub.2--. In certain
embodiments, R.sub.1, R.sub.3, and R.sub.5 each represent
independently for each occurrence hydrogen, halogen, hydroxyl,
amino, or C.sub.1-C.sub.6alkyl. In certain embodiments, R.sub.2 and
R.sub.4 are methoxy. In certain embodiments, R.sub.6 is
C(O)R.sub.8, and R.sub.8 is C.sub.1-C.sub.6alkyl. In certain
embodiments, R.sub.7 is alkyl. In certain embodiments, R.sub.8 and
R.sub.9 each represent independently hydrogen or
C.sub.1-C.sub.6alkyl. In certain embodiments, n is 0.
[0211] In certain embodiments, the Nec-3 related compound is
##STR00035##
or a pharmaceutically acceptable salt thereof.
[0212] In certain other embodiments, the Nec-3 related compound
is
##STR00036##
or a pharmaceutically acceptable salt thereof.
[0213] The Nec-3 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in Degterev et al. in Nature Chemical Biology, (2008), vol. 4,
313-321; and International Patent Application Publication No. WO
2007/075772, both of which is hereby incorporated by reference.
[0214] In certain embodiments, the necrostatin is a Nec-4 related
compound of Formula IV:
##STR00037##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0215] R.sub.1 is
##STR00038##
[0216] R.sub.2 and R.sub.3 each represent independently for each
occurrence hydrogen or methyl;
[0217] R.sub.4 represents independently for each occurrence
halogen, hydrogen, C.sub.1-C.sub.6alkyl, C.sub.2-C.sub.6alkenyl, or
C.sub.2-C.sub.4alkynyl;
[0218] R.sub.5 is C.sub.1-C.sub.4alkyl;
[0219] R.sub.6 is hydrogen, halogen, or --CN;
[0220] R.sub.7 is hydrogen or C.sub.1-C.sub.4alkyl;
[0221] R.sub.8 is C.sub.1-C.sub.6alkyl, or R.sub.8 taken together
with R.sub.9, when present, forms a carbocyclic ring;
[0222] R.sub.9 is hydrogen or C.sub.1-C.sub.6alkyl, or R.sub.9
taken together with R.sub.8 forms a carbocyclic ring;
[0223] R.sub.10 is hydrogen or C.sub.1-C.sub.6alkyl;
[0224] A is phenylene or a 5-6 membered heteroarylene;
[0225] X is N or --C(R.sub.9)--;
[0226] Y is N or --C(R.sub.10)--;
[0227] Z is S or O; and
[0228] m and n each represent independently 1, 2, or 3.
[0229] In certain embodiments, R.sub.1 is
##STR00039##
In certain other embodiments, R.sub.1 is
##STR00040##
In certain embodiments, R.sub.2 is hydrogen. In certain
embodiments, R.sub.3 is methyl. In certain other embodiments,
R.sub.3 is hydrogen. In certain embodiments, R.sub.4 is halogen,
such as fluorine or chlorine. In certain embodiments, R.sub.4 is
halogen. In certain embodiments, R.sub.5 is methyl or ethyl. In
certain embodiments, R.sub.6 is --CN. In certain embodiments, A is
phenylene. In certain embodiments, X is N. In certain embodiments,
Y is N. In certain embodiments, Z is S. In certain embodiments, A
is phenylene. In certain embodiments, R.sub.1 is
C.sub.1-C.sub.6alkyl, such as methyl. In certain embodiments, m is
1. In certain embodiments, n is 2.
[0230] In certain embodiments, the necrostatin is a Nec-4 related
compound of Formula IV-A:
##STR00041##
or a pharmaceutically acceptable salt thereof.
[0231] In certain embodiments, the necrostatin is a Nec-4 related
compound of Formula IV-B:
##STR00042##
or a pharmaceutically acceptable salt thereof.
[0232] The Nec-4 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in Teng et al. (2007) BIOORG MED CHEM LETT, 17: 6836-6840; and Teng
et al. (2008) BIOORG MED CHEM LETT, 18: 3219-3223, both of which
are incorporated herein by reference.
[0233] In certain embodiments, the necrostatin is a Nec-5 related
compound of Formula V:
##STR00043##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0234] A is a saturated or unsaturated 5-6 membered carbocyclic
ring;
[0235] X is a bond or C.sub.1-C.sub.4alkylene;
[0236] R.sub.1 is C.sub.1-C.sub.6alkyl, halogen, hydroxyl,
C.sub.1-C.sub.6alkoxyl, --N(R.sub.4).sub.2, --C(O)R.sub.4,
CO.sub.2R.sub.4, or C(O)N(R.sub.4).sub.2;
[0237] R.sub.2 is
##STR00044##
[0238] R.sub.3 is --C.sub.1-C.sub.6alkylene-CN, --CN,
C.sub.1-C.sub.6alkyl, or C.sub.2-C.sub.6alkenyl;
[0239] R.sub.4 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl;
[0240] R.sub.5 represents independently for each occurrence
C.sub.1-C.sub.6alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2;
[0241] B is a 5-6 membered heterocyclic or carbocylic ring; and
[0242] n and p each represent independently 0, 1, or 2.
[0243] In certain embodiments, X is a bond. In certain embodiments,
A is an unsaturated 6-membered carbocyclic ring. In certain
embodiments, R.sub.1 is C.sub.1-C.sub.6alkyl, halogen, hydroxyl, or
C.sub.1-C.sub.6alkoxyl. In certain embodiments, R.sub.2 is
##STR00045##
such as
##STR00046##
In certain embodiments, R.sub.3 is --C.sub.1-C.sub.6alkylene-CN,
such as --CH.sub.2--CN. In certain embodiments, R.sub.4 represents
independently for each occurrence hydrogen or C.sub.1-C.sub.6alkyl.
In certain embodiments, R.sub.5 represents independently for each
occurrence C.sub.1-C.sub.6alkyl, halogen, hydroxyl, or
C.sub.1-C.sub.6alkoxyl. In certain embodiments, B is a 5-6 membered
heterocyclic ring. In certain embodiments, n is 0. In certain
embodiments, p is 0.
[0244] In certain embodiments, the necrostatin is a Nec-5 related
compound of Formula V-A:
##STR00047##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0245] R.sub.1 is C.sub.1-C.sub.6alkyl, halogen, hydroxyl,
C.sub.1-C.sub.6alkoxyl, or --N(R.sub.4).sub.2;
[0246] R.sub.2 is
##STR00048##
[0247] R.sub.3 is --C.sub.1-C.sub.6alkylene-CN;
[0248] R.sub.4 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl;
[0249] R.sub.5 represents independently for each occurrence
C.sub.1-C.sub.6alkyl, halogen, hydroxyl, C.sub.1-C.sub.6alkoxyl,
--N(R.sub.4).sub.2, --C(O)R.sub.4, CO.sub.2R.sub.4, or
C(O)N(R.sub.4).sub.2;
[0250] B is a 5-6 membered heterocyclic or carbocylic ring; and
[0251] n and p each represent independently 0, 1, or 2.
[0252] In certain embodiments, the Nec-5 compound is
##STR00049##
or a pharmaceutically acceptable salt thereof.
[0253] The Nec-5 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in Degterev et al. in Nature Chemical Biology, (2008), vol. 4,
313-321; and International Patent Application Publication No. WO
2008/045406, both of which is hereby incorporated by reference.
[0254] In certain embodiments, the necrostatin is a Nec-7 related
compound of Formula VII:
##STR00050##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0255] R.sub.1, R.sub.2, and R.sub.3 each represent independently
hydrogen or C.sub.1-C.sub.4alkyl;
[0256] R.sub.4 is
##STR00051##
[0257] R.sub.5 and R.sub.6 each represent independently for each
occurrence halogen, C.sub.1-C.sub.6alkyl, hydroxyl,
C.sub.1-C.sub.6alkoxyl, --N(R.sub.7).sub.2, --NO.sub.2,
--S--C.sub.1-C.sub.6alkyl, --S-aryl,
--SO.sub.2--C.sub.1-C.sub.6alkyl, --SO.sub.2-aryl, --C(O)R.sub.7,
--CO.sub.2R.sub.7, --C(O)N(R.sub.7).sub.2, heterocycloalkyl, aryl,
or heteroaryl;
[0258] R.sub.7 represents independently for each occurrence
hydrogen, C.sub.1-C.sub.6alkyl, aryl, or aralkyl; or two
occurrences of R.sub.7 attached to the same nitrogen atom are taken
together with the nitrogen atom to which they are attached to form
a 3-7 membered heterocyclic ring;
[0259] A is a 5-6 membered heterocyclic ring; and
[0260] p is 0, 1, or 2.
[0261] In certain embodiments, R.sub.1 is hydrogen. In certain
embodiments, R.sub.2 is hydrogen. In certain embodiments, R.sub.3
is hydrogen. In certain embodiments, R.sub.4 is
##STR00052##
In certain embodiments, R.sub.5 is halogen, C.sub.1-C.sub.6alkyl,
hydroxyl, C.sub.1-C.sub.6alkoxyl, or --N(R.sub.7).sub.2. In certain
other embodiments, R.sub.5 is halogen, such as fluorine or
chlorine. In certain embodiments, p is 0. In certain other
embodiments, R.sub.4 is
##STR00053##
such as
##STR00054##
In certain embodiments, the Nec-7 related compound is
##STR00055##
or a pharmaceutically acceptable salt thereof.
[0262] The Nec-7 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
in Zheng et al. in BIOORG MED CHEM LETT, 2008, vol. 18, 4932-4935,
which is incorporated herein by reference.
[0263] In certain embodiments, the necrostatin is a Nec-7 related
compound of Formula VIII:
##STR00056##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0264] each X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, and
X.sup.6 is selected, independently, from N or CR.sup.X1;
[0265] each Y.sup.1, Y.sup.2, and Y.sup.3 is selected,
independently, from O, S, NR.sup.Y1, or CR.sup.Y2R.sup.Y3;
[0266] each Z.sup.1 and Z.sup.2 is selected, independently, from O,
S, or NR.sup.Z;
[0267] each R.sup.Y1 and R.sup.Z1 is selected, independently, from
H, optionally substituted C.sub.1-6alkyl, optionally substituted
C.sub.2-6alkenyl, optionally substituted C.sub.2-6alkynyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, --C(.dbd.O)R.sup.5A, --C(.dbd.O)OR.sup.5A, or
--C(.dbd.O)NR.sup.5AR.sup.6A;
[0268] each R.sup.X1, R.sup.Y2, and R.sup.Y3 is selected,
independently, from H, halogen, CN, NC, NO.sub.2, N.sub.3,
OR.sup.3, SR.sup.3, NR.sup.3R.sup.4, --C(.dbd.O)R.sup.5A,
--C(.dbd.O)OR.sup.5A, --C(.dbd.O)NR.sup.5AR.sup.6A,
--S(.dbd.O)R.sup.5A, --S(.dbd.O).sub.2R.sup.5A,
--S(.dbd.O).sub.2OR.sup.5A, --S(.dbd.O).sub.2NR.sup.5AR.sup.6A,
optionally substituted C.sub.1-6alkyl, optionally substituted
C.sub.2-6alkenyl, optionally substituted C.sub.2-6alkynyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, or optionally
substituted heteroaryl;
[0269] each R.sup.1, R.sup.2 R.sup.5A, R.sup.5B, R.sup.6A, and
R.sup.6B is selected from H, optionally substituted C.sub.1-6
alkyl, optionally substituted C.sub.2-6alkenyl, optionally
substituted C.sub.2-6alkynyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
or optionally substituted heteroaryl; or R.sup.5A and R.sup.6A, or
R.sup.5B and R.sup.6B combine to form a heterocyclyl; and
[0270] each R.sup.3 and R.sup.4 is selected from H, optionally
substituted C.sub.1-6 alkyl, optionally substituted cycloalkyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, --C(.dbd.O)R.sup.5B,
--C(.dbd.S)R.sup.5B, --C(.dbd.NR.sup.6B)R.sup.5B,
--C(.dbd.O)OR.sup.5B, --C(.dbd.O)NR.sup.5BR.sup.6B,
--S(.dbd.O)R.sup.5B, --S(.dbd.O).sub.2R.sup.5B,
--S(.dbd.O).sub.2OR.sup.5B, or --S(.dbd.O).sub.2NR.sup.5BR.sup.6B.
In certain embodiments, when R.sup.1 is H, X.sup.1, X.sup.2, and
X.sup.4 are each CH, X.sup.3, X.sup.5, and X.sup.6 are each N,
Y.sup.1 and Y.sup.3 are each S, Y.sup.2 is NH, Z.sup.1 is NH, and
Z.sup.2 is O, then R.sup.2 is not 4-fluorophenyl.
[0271] In certain embodiments, the necrostatin is a Nec-7 related
compound of Formula VIII-A:
##STR00057##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0272] X.sup.1, X.sup.2, X.sup.4, X.sup.5, R.sup.1, Y.sup.2, and
R.sup.Z1 are as defined for Formula (VIII);
[0273] each R.sup.2A, R.sup.2B, R.sup.2C, R.sup.2D, and R.sup.2E is
selected, independently, from H, halogen, optionally substituted
C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl,
optionally substituted C.sub.2-6 alkynyl, optionally substituted
cycloalkyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl, CN, NC,
NO.sub.2, N.sub.3, OR.sup.7, SR.sup.7, S(.dbd.O)R.sup.12,
S(.dbd.O).sub.2R.sup.12, S(.dbd.O)OR.sup.12,
S(.dbd.O).sub.2OR.sup.12, NR.sup.7R.sup.8, C(.dbd.O)R.sup.12,
C(.dbd.O)OR.sup.12, C(.dbd.O)NR 1R.sup.13, C(.dbd.S)R.sup.12,
C(.dbd.S)OR.sup.12, C(.dbd.S)NR.sup.12R.sup.13,
C(.dbd.NR.sup.9)R.sup.12, C(.dbd.NR.sup.9)OR.sup.12, or
C(.dbd.NR.sup.9)NR.sup.12R.sup.13, or R.sup.2A and R.sup.2B,
R.sup.2B and R.sup.2CR.sup.2C and R.sup.2D, or R.sup.2D and
R.sup.2E combine to form an optionally substituted cycloalkyl or an
optionally substituted heterocyclyl;
[0274] each R.sup.7, R.sup.8, and R.sup.9 is selected,
independently, from H, optionally substituted C.sub.1-6 alkyl,
optionally substituted cycloalkyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, S(.dbd.O)R.sup.10, S(.dbd.O).sub.2R.sup.10,
C(.dbd.O)R.sup.10, C(.dbd.O)OR.sup.10, C(.dbd.O)NR.sup.10R.sup.11,
C(.dbd.S)R.sup.10, C(.dbd.S)OR.sup.10, C(.dbd.S)NR.sup.10R.sup.11,
C(.dbd.NR.sup.14)R.sup.10, C(.dbd.NR.sup.14)OR.sup.10, or
C(.dbd.NR.sup.14)NR.sup.10R.sup.11, or R.sup.7 and R.sup.8 combine
to form an optionally substituted heterocyclyl; and each R.sup.10,
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 is selected,
independently, from H, optionally substituted C.sub.1-6alkyl,
optionally substituted C.sub.2-6alkenyl, optionally substituted
C.sub.2-6alkynyl, optionally substituted cycloalkyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl, or R.sup.10 and R.sup.1 or
R.sup.12 and R.sup.13 combine to form an optionally substituted
heterocyclyl.
[0275] In certain embodiments, each R.sup.2A, R.sup.2B, R.sup.2C,
R.sup.2D, and R.sup.2E is selected, independently, from H, halogen,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, cycloalkyl,
heterocyclyl, aryl, or heteroaryl.
[0276] In certain embodiments, the necrostatin is a Nec-7 related
compound selected from:
##STR00058## ##STR00059## ##STR00060##
and pharmaceutically acceptable salts thereof.
[0277] The Nec-7 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
International Patent Application Publication No. WO 2010/075290,
which is hereby incorporated by reference.
[0278] In certain embodiments, the necrostatin is a Nec-4 related
compound of Formula IX:
##STR00061##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0279] X.sub.1 and X.sub.2 are, independently, N or CR.sup.4;
[0280] X.sub.3 is selected from O, S, NR.sup.5, or
--(CR.sup.5).sub.2;
[0281] Y is selected from C(O) or CH.sub.2; and
[0282] Z is (CR.sup.6R.sup.7).sub.n;
[0283] R.sup.1 is selected from H, halogen, optionally substituted
C.sub.1-6alkyl, or optionally substituted C.sub.1-6cycloalkyl, or
optionally substituted aryl;
[0284] R.sup.2 is selected from H or optionally substituted
C.sub.1-6alkyl;
[0285] R.sup.3 is optionally substituted aryl;
[0286] each R.sup.4 is selected from H, halogen, carboxamido,
nitro, cyano, optionally substituted C.sub.1-6alkyl, or optionally
substituted aryl;
[0287] R.sup.5 is selected from H, halogen, optionally substituted
C.sub.1-6alkyl, or optionally substituted aryl;
[0288] each R.sup.6 and R.sup.7 is, independently, selected from H,
optionally substituted C.sub.1-6alkyl, or aryl; and
[0289] n is 0, 1, 2, or 3. In certain embodiments, when X.sub.1 and
X.sub.2 are N, X.sub.3 is S, Y is C(O), Z is CH.sub.2, R.sup.2 is
H, and R.sup.3 is 2-chloro-6-fluoro-phenyl, then R.sup.1 is not
methyl.
[0290] In certain embodiments, the necrostatin is a Nec-4 related
compound of Formula IX-A:
##STR00062##
or a pharmaceutically acceptable salt, ester, or prodrug thereof,
wherein:
[0291] R.sup.1, R.sup.2, R.sup.3, R.sup.6 and R.sup.7 are as
defined in Formula (IX).
[0292] In certain embodiments, the necrostatin is a Nec-4 related
compound selected from:
##STR00063## ##STR00064##
and pharmaceutically acceptable salts thereof.
[0293] The Nec-4 related compounds described above can be prepared
based on synthetic procedures described in the literature, such as
U.S. Patent Application Publication No. 2009/0099242, which is
hereby incorporated by reference.
[0294] The term "alkyl" is art-recognized, and includes saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In certain embodiments, a straight chain or branched chain
alkyl has about 10 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.10 for straight chain, C.sub.3-C.sub.10 for branched
chain), and alternatively, 5, 4, 3, 2 or 1 carbon atoms in its
backbone. Likewise, cycloalkyls have from about 3 to about 10
carbon atoms in their ring structure, and alternatively about 5, 6
or 7 carbons in the ring structure. Exemplary alkyl groups include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, cyclopropyl, and cyclobutyl.
[0295] The terms "alkoxyl" or "alkoxy" are art-recognized and refer
to an alkyl group, as defined above, having an oxygen radical
attached thereto. Representative alkoxyl groups include methoxy,
ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two
hydrocarbons covalently linked by an oxygen. Accordingly, the
substituent of an alkyl that renders that alkyl an ether is or
resembles an alkoxyl, such as may be represented by one of
--O-alkyl, --O-alkenyl, or --O-alkynyl. The term "alkylene" refers
to a diradical of an alkyl group. An exemplary alkylene group is
--CH.sub.2CH.sub.2--.
[0296] The term "aralkyl" refers to an alkyl group substituted with
an aryl group.
[0297] The term "heteroaralkyl" refers to an alkyl group
substituted with a heteroaryl group.
[0298] The term "alkenyl" refers to an unsaturated straight or
branched hydrocarbon having at least one carbon-carbon double bond,
such as a straight or branched group of 2-12, 2-10, or 2-6 carbon
atoms, referred to herein as C.sub.2-C.sub.12alkenyl,
C.sub.2-C.sub.10alkenyl, and C.sub.2-C.sub.6alkenyl, respectively.
Exemplary alkenyl groups include, but are not limited to, vinyl,
allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl,
hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,
4-(2-methyl-3-butene)-pentenyl, etc.
[0299] The term "alkynyl" as used herein refers to an unsaturated
straight or branched hydrocarbon having at least one carbon-carbon
triple bond, such as a straight or branched group of 2-12, 2-8, or
2-6 carbon atoms, referred to herein as C.sub.2-C.sub.12alkynyl,
C.sub.2-C.sub.10alkynyl, and C.sub.2-C.sub.6alkynyl, respectively.
Exemplary alkynyl groups include, but are not limited to, ethynyl,
propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,
4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl,
etc.
[0300] The term "aryl" is art-recognized and refers to a
carbocyclic aromatic group. Representative aryl groups include
phenyl, naphthyl, anthracenyl, and the like. Unless specified
otherwise, the aromatic ring may be substituted at one or more ring
positions with, for example, halogen, azide, alkyl, aralkyl,
alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,
sulfhydryl, imino, amido, carboxylic acid, --C(O)alkyl,
--CO.sub.2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl,
sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl,
heteroaryl, --CF.sub.3, --CN, or the like. The term "aryl" also
includes polycyclic ring systems having two or more carbocyclic
rings in which two or more carbons are common to two adjoining
rings (the rings are "fused rings") wherein at least one of the
rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,
cycloalkenyls, cycloalkynyls, and/or aryls.
[0301] In certain embodiments, the aromatic group is not
substituted, i.e., it is unsubstituted.
[0302] The term "phenylene" refers to a multivalent radical (e.g.,
a divalent or trivalent radical) of benzene. To illustrate, a
divalent valent radical of benzene is illustrated by the
formula
##STR00065##
[0303] The terms "heterocyclyl" or "heterocyclic group" are
art-recognized and refer to saturated, partially unsaturated, or
aromatic 3- to 10-membered ring structures, alternatively 3- to
7-membered rings, whose ring structures include one to four
heteroatoms, such as nitrogen, oxygen, and sulfur. Heterocycles may
also be mono-, bi-, or other multi-cyclic ring systems. A
heterocycle may be fused to one or more aryl, partially
unsaturated, or saturated rings. Heterocyclyl groups include, for
example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl,
dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl,
imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl,
piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl,
pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl,
tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl,
tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl,
thiopyranyl, xanthenyl, lactones, lactams such as azetidinones and
pyrrolidinones, sultams, sultones, and the like. Unless specified
otherwise, the heterocyclic ring is optionally substituted at one
or more positions with substituents such as alkanoyl, alkoxy,
alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl,
azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester,
ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl,
hydroxyl, imino, ketone, nitro, phosphate, phosphonato,
phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and
thiocarbonyl. In certain embodiments, the heterocyclcyl group is
not substituted, i.e., it is unsubstituted.
[0304] The term "heteroaryl" is art-recognized and refers to
aromatic groups that include at least one ring heteroatom. In
certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring
heteroatoms. Representative examples of heteroaryl groups include
pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl,
triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and
pyrimidinyl, and the like. Unless specified otherwise, the
heteroaryl ring may be substituted at one or more ring positions
with, for example, halogen, azide, alkyl, aralkyl, alkenyl,
alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl,
imino, amido, carboxylic acid, --C(O)alkyl, --CO.sub.2alkyl,
carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,
ketone, aldehyde, ester, heterocyclyl, aryl, --CF.sub.3, --CN, or
the like. The term "heteroaryl" also includes polycyclic ring
systems having two or more rings in which two or more carbons are
common to two adjoining rings (the rings are "fused rings") wherein
at least one of the rings is heteroaromatic, e.g., the other cyclic
rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or
aryls.
[0305] The term "heteroarylene" refers to a multi-valent (e.g.,
di-valent or trivalent) aromatic group that comprises at least one
ring heteroatom. An exemplary "heteroarylene" is pyridinylene,
which is a multi-valent radical of pyridine. For example, a
divalent radical of pyridine is illustrated by the formula
##STR00066##
[0306] The terms ortho, meta and para are art-recognized and refer
to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For
example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene
are synonymous.
[0307] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
may be represented by the general formula:
##STR00067##
wherein R.sup.50 and R.sup.51 each independently represent
hydrogen, alkyl, alkenyl, or --(CH.sub.2).sub.m--R.sup.61; or
R.sup.50 and R.sup.51, taken together with the N atom to which they
are attached complete a heterocycle having from 4 to 8 atoms in the
ring structure; wherein R.sup.61 is aryl, cycloalkyl, cycloalkenyl,
a heterocycle or a polycycle; and m is zero or an integer in the
range of 1 to 8. In certain embodiments, R.sup.50 and R.sup.51 each
independently represent hydrogen or alkyl.
[0308] The term "amide" or "amido" as used herein refers to a
radical of the form --R.sub.aC(O)N(R.sub.b)--,
--R.sub.aC(O)N(R.sub.b)R.sub.c--, --C(O)NR.sub.bR.sub.c, or
--C(O)NH.sub.2, wherein R.sub.a, R.sub.b and R.sub.c are each
independently selected from alkoxy, alkyl, alkenyl, alkynyl, amide,
amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether,
formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen,
hydroxyl, ketone, and nitro. The amide can be attached to another
group through the carbon, the nitrogen, R.sub.b, R.sub.c, or
R.sub.a. The amide also may be cyclic, for example R.sub.b and
R.sub.c, R.sub.a and R.sub.b, or R.sub.a and R.sub.c may be joined
to form a 3- to 12-membered ring, such as a 3- to 10-membered ring
or a 5- to 6-membered ring. The term "carboxamido" refers to the
structure --C(O)NR.sub.bR.sub.c.
[0309] The term "sulfonamide" or "sulfonamido" as used herein
refers to a radical having the structure
--N(R.sub.r)--S(O).sub.2--R.sub.s-- or
--S(O).sub.2--N(R.sub.r)R.sub.s, where R.sub.r, and R.sub.s can be,
for example, hydrogen, alkyl, aryl, cycloalkyl, and heterocyclyl.
Exemplary sulfonamides include alkylsulfonamides (e.g., where
R.sub.s is alkyl), arylsulfonamides (e.g., where R.sub.s is aryl),
cycloalkyl sulfonamides (e.g., where R.sub.s is cycloalkyl), and
heterocyclyl sulfonamides (e.g., where R.sub.s is heterocyclyl),
etc.
[0310] The term "sulfonyl" as used herein refers to a radical
having the structure R.sub.uSO.sub.2--, where R.sub.u can be alkyl,
aryl, cycloalkyl, and heterocyclyl, e.g., alkylsulfonyl. The term
"alkylsulfonyl" as used herein refers to an alkyl group attached to
a sulfonyl group.
[0311] The symbol "" indicates a point of attachment.
[0312] Unless specified otherwise, the term "optionally
substituted" as used herein means that the specified group may be
substituted at one, two or more positions with, for example,
halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,
carboxylic acid, --C(O)alkyl, --CO.sub.2alkyl, carbonyl, carboxyl,
alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde,
ester, heterocyclyl, heteroaryl, --CF.sub.3, --CN, or the like.
[0313] As used herein, the term "therapeutically effective amount"
is understood to mean the amount of an active ingredient, for
example, a necrostatin (e.g., necrostatin-1 or necrostatin-4)
and/or a pan-caspase inhibitor (e.g., Z-VAD or IDN-6556) that is
sufficient to reduce, minimize or eliminate the death of
photoreceptor and/or RPE cells associated with certain ocular
disorders described herein. The compounds of the invention are
administered in amounts effective at, e.g., reducing the death of
photoreceptor and/or RPE cells, increasing efficacy compared to
monotherapy with either drug alone, preserving or improving vision,
preserving or improving visual function, and/or preventing vision
loss. It is understood that preserving vision or visual function,
includes stabilizing vision or visual function and/or slowing the
decline of vision or visual function prior to treatment.
[0314] As used herein, "pharmaceutically acceptable" or
"pharmacologically acceptable" mean molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or to a human, as
appropriate. The term, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0315] Disclosed herein is a method of preserving the visual
function of an eye of a subject with an ocular condition, wherein a
symptom of the ocular condition is the loss of photoreceptor cell
viability in the retina of the eye with the condition. The method
comprises (a) administering to the eye of the subject an effective
amount of a necrostatin and an effective amount of an apoptosis
inhibitor thereby preserving the viability of the photoreceptor
cells disposed within the retina of the eye, and (b) measuring the
visual function (e.g., visual acuity) of the eye after the
administration of the necrosis inhibitor and the apoptosis
inhibitor. After administration of the necrosis inhibitor and the
apoptosis inhibitor the visual function of the eye may be preserved
or improved relative to the visual function of the eye prior to
administration of the necrosis inhibitor and the apoptosis
inhibitor.
[0316] Also disclosed is a method of preserving the visual function
of an eye of a subject with an ocular condition wherein a symptom
of the eye is the loss of photoreceptor cell and/or RPE cell
viability in the retina of the eye. The method comprises reducing
the production and/or activity of a RIP-1 kinase and/or a RIP-3
kinase in the eye to preserve the viability of the photoreceptor
cells and/or RPE cells disposed within the eye. The reduction in
the production and/or activity of the RIP-1 kinase and/or RIP-3
kinase may be achieved by administering an effective amount of a
necrosis inhibitor (e.g., RIPK inhibitor, e.g., a necrostatin). The
reduction in the production and/or activity of the RIP-1 kinase
and/or RIP-3 kinase may be direct (e.g., the necrosis inhibitor
modulates the production and/or activity the RIP-1 kinase and/or
RIP-3 kinase directly) or indirect (e.g., the necrosis inhibitor
acts upstream of the RIP-1 kinase and/or RIP-3 kinase but the
administration of which indirectly modulates the production and/or
activity of the RIP-1 kinase and/or RIP-3 kinase). Visual function
of the eye may be measured before and/or after the administration
of the necrosis inhibitor that directly or indirectly reduces the
production and/or activity of a RIP-1 kinase and/or RIP-3 kinase.
After administration of the necrosis inhibitor the visual function
of the eye may be preserved or improved relative to the visual
function of the eye prior to administration of the necrosis
inhibitor.
[0317] In each of the foregoing methods, the ocular condition,
wherein a symptom of the condition is the loss of photoreceptor
cell viability in the retina of the eye, includes but is not
limited to AMD (e.g., dry AMD or neovascular AMD), RP and allied
diseases (i.e., diseases in the same spectrum, for example, Usher's
Syndrome, Cone-Rod dystrophy, and Bardet-Biedl Syndrome), retinal
detachment, macular edema (e.g, macular edema caused by vein
occlusion, diabetes and intraocular inflammation), diabetic
retinopathy, central areolar choroidal dystrophy, BEST disease,
adult vitelliform disease, pattern dystrophy, myopic degeneration,
central serous retinopathy, Stargardt's disease, Cone-Rod
dystrophy, North Carolina dystrophy, infectious retinitis,
inflammatory retinitis, uveitis, toxic retinitis, and light induced
toxicity.
[0318] Also disclosed is a method of preserving the viability of
photoreceptor cells disposed within a retina of a mammalian eye
affected by AMD, RP, macular edema, central areolar choroidal
dystrophy, diabetic retinopathy, BEST disease, adult vitelliform
disease, pattern dystrophy, myopic degeneration, central serous
retinopathy, Stargardt's disease, Cone-Rod dystrophy, North
Carolina dystrophy, infectious retinitis, inflammatory retinitis,
uveitis, toxic retinitis and light-induced toxicity. The method
comprises administering a necrosis inhibitor and/or an apoptosis
inhibitor to the eye in which a region of the retina has been
affected in amounts sufficient to preserve the viability of
photoreceptor cells disposed within the region of the affected
retina.
[0319] Also disclosed is a method of preserving the viability of
RPE cells of a subject with an ocular condition, wherein a symptom
of the ocular condition is the loss of RPE cell in the retina of an
eye with the condition. The method comprises administering a
necrosis inhibitor and/or an apoptosis inhibitor to the eye in
which a region of the retina has been affected in amounts
sufficient to preserve the viability of the RPE cells.
[0320] In each of the foregoing methods, the ocular condition,
wherein a symptom of the condition is the loss of RPE cell
viability in the retina of the eye, includes but is not limited to
AMD (e.g., dry AMD or neovascular AMD), BEST disease, Stargardt's
disease, uveitis, adult foveomacular dystrophy, fundus
flavimaclatus, myopic degeneration, multiple evanescent white dot
syndrome, serpiginous choroidopathy, AMPPE, and other uveitis
disorders.
[0321] Also disclosed is a method of preserving the viability of
photoreceptor cells disposed within a retina of a mammalian eye
following retinal detachment. The method comprises administering a
necrostatin and an apoptosis inhibitor to the eye in which a region
of the retina has been detached in amounts sufficient to preserve
the viability of photoreceptor cells disposed within the region of
the detached retina. In certain embodiments, when the only
necrostatin administered is necrostatin-1, the region is exposed to
a final concentration of necrostatin in the eye of greater than
about 100 .mu.M. The retinal detachment can be rhegmatogenous
retinal detachment, tractional retinal detachment, or serous
retinal detachment.
[0322] As discussed above, the prevention of photoreceptor death
following retinal detachment by co-administration of an apoptotic
inhibitor and a necrostatin, e.g., necrostatin-1, at concentrations
that exceed 100 .mu.M was surprising because it had been believed
that concentrations of necrostatin-1 exceeding 100 .mu.M were toxic
and, therefore, could not be administered at such a dosage. It was
an unexpected finding that the combination of a necrostatin, e.g.,
necrostatin-1, and a pan-caspase inhibitor, e.g., Z-VAD, achieved a
synergistic effect in reducing photoreceptor cell death following
retinal detachment compared to either drug alone.
[0323] Unless specified, the necrostatin can be administered to
give a final concentration of greater than about 30 .mu.M, for
example, in the range of about 30 .mu.M to about 1000 .mu.M. In
certain embodiments, the necrostatin can be administered in an
amount sufficient to give a final concentration of necrostatin in
the eye of greater than about 30 .mu.M. For example, the
necrostatin may be administered in an amount sufficient to give a
final concentration of necrostatin in the eye in the range from
about 30 .mu.M to about 1000 .mu.M, 50 .mu.M to about 1000 .mu.M,
80 .mu.M to about 1000 .mu.M, about 100 .mu.M to about 1000 .mu.M,
about 150 .mu.M to about 1000 .mu.M, from about 200 .mu.M to about
800 .mu.M, or from about 200 .mu.M to about 600 .mu.M. In certain
embodiments, the necrostatin is administered in an amount
sufficient to give a final concentration of necrostatin in the eye
of about 400 .mu.M.
[0324] The apoptosis inhibitor, for example, the pan-caspase
inhibitor, can be administered in an amount sufficient to give a
final concentration of the inhibitor in the eye of greater than
about 40 .mu.M, for example, in the range of about 40 .mu.M to
about 500 .mu.M. For example, the apoptosis inhibitor can be
administered in an amount sufficient to give a final concentration
of the inhibitor in the eye of greater than about 60 .mu.M, 80
.mu.M, or 100 .mu.M. For example, the apoptosis inhibitor can be
administered in an amount sufficient to give a final concentration
of the inhibitor in the eye in the range from about 80 .mu.M to
about 500 .mu.M, 100 .mu.M to about 500 .mu.M, 125 .mu.M to about
500 .mu.M, 150 .mu.M to about 500 .mu.M or from about 200 .mu.M to
about 400 .mu.M. In certain embodiments, apoptosis inhibitor (e.g.,
the pan-caspase inhibitor) is administered in an amount sufficient
to give a final concentration of the inhibitor in the eye of about
300 .mu.M.
[0325] In view of the fact that the volume of the eye in a given
subject is known (for example, typical human eye contains 4 to 6 mL
of fluid (humor)) it is possible to calculate the dosage of the
necrostatin and/or the pan-caspase inhibitor to be administered to
give the therapeutically effective concentrations noted above. For
example, from about 0.035 mg to about 2 mg of necrostatin-1 and
from about 0.05 mg to about 1.5 mg of a pan-caspase inhibitor can
be administered to achieve the concentrations noted above.
[0326] In certain embodiments, from about 0.025 mg to about 4 mg,
from about 0.035 mg to about 2 mg, from about 0.05 mg to about 2
mg, from about 0.1 mg to about 2 mg, from about 0.2 mg to about 1
mg, or from about 0.2 mg to about 0.8 mg of the necrosis inhibitor
(e.g., a necrostatin) can be administered locally to the eye of a
mammal. In one embodiment, 0.5 mg of necrostatin is administered
locally to the eye of a mammal. In certain other embodiments, from
about 0.05 mg to about 2 mg, from about 0.2 mg to about 2 mg, from
about 0.05 mg to about 1.5 mg, from about 0.15 mg to about 1.5 mg,
from about 0.4 mg to about 1 mg, or from about 0.5 mg to about 0.8
mg of an apoptosis inhibitor (e.g., a pan-caspase inhibitor, e.g.,
Z-VAD) can be administered locally to the eye of a mammal. In
certain embodiments, about 0.7 mg of a pan-caspase inhibitor, e.g.,
Z-VAD, is administered locally to the eye of a mammal.
[0327] It is understood that one or more of a necrosis inhibitor,
one or more of an apoptosis inhibitor, or one or more of a necrosis
inhibitor and one or more of an apoptosis inhibitor can be
administered to the eye in which a region of the retina has been
affected in amounts sufficient to preserve the viability of
photoreceptor cells disposed within the region of the affected
retina.
[0328] In certain embodiments, the necrosis inhibitor is a
necrostatin, for example, necrostatin-1, a necrostatin-2, a
necrostatin-4, a necrostatin-5, and a necrostatin-7. One or more of
these necrosis inhibitors can be administered with one or more of
the apoptosis inhibitors (e.g., IDN-6556) listed below.
Furthermore, it is contemplated that one or more of the
necrostatins shown by Formula I, I-A, I-B, I-C, I-D, I-E, I-F, I-G,
II, II-A, III, IV, IV-A, IV-B, V, V-A, VII, VIII, VIII-A, IX, or
IX-A can be administered with one or more of the apoptosis
inhibitors (e.g., IDN-6556 or IDN-6734) listed below.
[0329] In certain embodiment, the necrosis inhibitor reduces the
production and/or activity of a RIP-1 kinase and/or a RIP-3 kinase.
RIP kinase inhibitors (e.g., RIP-1 kinase and/or RIP-3 kinase
inhibitors) as disclosed herein may further include RNAs, including
small inhibitory RNAs (siRNAs) and short hairpin RNAs (shRNAs).
Methods for designing and synthesizing siRNAs and shRNAs are well
known in the art. Exemplary RIP-1 kinase inhibitors include, for
example, a pSIREN-RIP-1 shRNA construct which targets RIP-1 kinase
as disclosed in Kaiser et al. (2008) JOURNAL OF IMMUNOLOGY
181:6427-6434. Exemplary RIP-3 kinase inhibitors include, for
example, sc-61482-SH and sc-135170 available from Santa Cruz
Biotechnology. In another example, RIP kinase inhibitors (e.g.,
RIP-1 kinase and/or RIP-3 kinase inhibitors) as disclosed herein
may include inhibitor of apoptosis proteins (IAPs), active
fragments thereof, and nucleic acids encoding the same. It is well
established that IAPs inhibit RIP-1 kinase by functioning as a E3
ligase for RIP-1 kinase (see, for example, Vanlangenakker et al.
(2010) CELL DEATH AND DIFFERENTIATION 18:656-665).
[0330] In certain embodiments, the one or more apoptosis inhibitors
may include a pan-caspase inhibitor. The pan-caspase inhibitor can
be Z-VAD (i.e., Z-Val-Ala-Asp(OMe)-CH.sub.2F*), IDN-6556 available
from Conatus Pharmaceuticals (i.e.,
(3-{2-[(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2-
,3,5,6-tetrafluoro-phenoxy)-pentanoic acid)
(3-{2-[(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2-
,3,5,6-tetrafluoro-phenoxy)-pentanoic acid), IDN-6734 available
from Conatus Pharmaceuticals, VX-799 available from Vertex
Pharmaceuticals, MX1013 and MX2060 derivatives available from Maxim
Pharmaceuticals, M-920 available from Merck-Frosst, small-molecule
compounds available from Gemin X Pharmaceuticals, RGD peptides from
Merck-Frost and Maxim Pharmaceuticals, or any other known
pan-caspase inhibitor.
[0331] Alternatively, the pan-caspase inhibitor can be a cocktail
of caspase inhibitors including two or more specific caspase
inhibitors (e.g., synthetic caspase inhibitors) such as a caspase 1
inhibitor, a caspase 2 inhibitor, a caspase 3 inhibitor, a caspase
4 inhibitor, a caspase 5 inhibitor, a caspase 6 inhibitor, a
caspase 7 inhibitor, a caspase 8 inhibitor, and a caspase 9
inhibitor. It is contemplated that one or more of the pan-caspase
inhibitors may be used in combination with one or more necrostatins
(e.g., necrostain-1 and/or necrostatin-4).
[0332] Exemplary synthetic caspase 1 inhibitors, include, for
example, Ac--N-Me-Tyr-Val-Ala-Asp-aldehyde,
Ac-Trp-Glu-His-Asp-aldehyde, Ac-Tyr-N-Me-Val-Ala-N-Me-Asp-aldehyde,
Ac-Tyr-Val-Ala-Asp-Aldehyde, Ac-Tyr-Val-Ala-Asp-chloromethylketone,
Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone,
Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetol,
Ac-Tyr-Val-Lys-Asp-aldehyde,
Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone,
biotinyl-Tyr-Val-Ala-Asp-chloromethylketone,
Boc-Asp(OBzl)-chloromethylketone,
ethoxycarbonyl-Ala-Tyr-Val-Ala-Asp-aldehyde (pseudo acid),
Z-Asp-2,6-dichlorobenzoyloxymethylketone,
Z-Asp(OlBu)-bromomethylketone,
Z-Tyr-Val-Ala-Asp-chloromethylketone,
Z-Tyr-Val-Ala-DL-Asp-fluoromethlyketone,
Z-Val-Ala-DL-Asp-fluoromethylketone, and
Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone, all of which can be
obtained from Bachem Bioscience Inc., PA. Other exemplary caspase 1
inhibitors include, for example, Z-Val-Ala-Asp-fluoromethylketone,
biotin-X-Val-Ala-Asp-fluoromethylketone, Ac-Val-Ala-Asp-aldehyde,
Boc-Asp-fluoromethylketone,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Pro-Tyr-Val-Al-
a-Asp-aldehyde (SEQ ID NO: 1),
biotin-Tyr-Val-Ala-Asp-fluoroacyloxymethylketone,
Ac-Tyr-Val-Ala-Asp-acyloxymethylketone, Z-Asp-CH2-DCB, and
Z-Tyr-Val-Ala-Asp-fluoromethylketone, all of which are available
from Calbiochem, IDN-11104 available from Conatus Pharmaceuticals,
and VX-740 and VX-756 available from Vertex Pharmaceuticals.
[0333] Exemplary synthetic caspase 2 inhibitors, include, for
example, Ac-Val-Asp-Val-Ala-Asp-aldehyde, which can be obtained
from Bachem Bioscience Inc., PA, and
Z-Val-Asp-Val-Ala-Asp-fluoromethylketone, which can be obtained
from Calbiochem, CA.
[0334] Exemplary synthetic caspase 3 precursor protease inhibitors
include, for example, Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid) and
Ac-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) which can be obtained
from Bachem Bioscience Inc., PA. Exemplary synthetic caspase 3
inhibitors include, for example, Ac-Asp-Glu-Val-Asp-aldehyde,
Ac-Asp-Met-Gin-Asp-aldehyde, biotinyl-Asp-Glu-Val-Asp-aldehyde,
Z-Asp-Glu-Val-Asp-chloromethylketone,
Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone, and
Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone which can be obtained from
Bachem Bioscience Inc., PA. Other exemplary caspase 3 inhibitors
include, for example,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Gl-
u-Val-Asp-aldehyde (SEQ ID NO: 2),
biotin-X-Asp-Glu-Val-Asp-fluoromethylketone,
Ac-Asp-Glu-Val-Asp-chloromethylketone, all of which are available
from Calbiochem. Another exemplary caspase 3 inhibitor includes,
the caspase 3 inhibitor
N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyk-
etone (z-Asp-Glu-Val-Asp-fmk), which is available from Enzyme
Systems Products. Additional exemplary caspase 3 inhibitors include
M-826 and M-791 available from Merck-Frosst, Immunocasp-3,
Ad-G/iCasp3, and PEF-F8-CP3.
[0335] Exemplary synthetic caspase 4 inhibitors include, for
example, Ac-Leu-Glu-Val-Asp-aldehyde and
Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone, which can be obtained from
Bachem Bioscience Inc., PA, and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Gl-
u-Val-Pro-aldehyde (SEQ ID NO: 3), which can be obtained from
Calbiochem, CA.
[0336] Exemplary synthetic caspase 5 inhibitors include, for
example, Z-Trp-His-Glu-Asp-fluoromethylketone, which can be
obtained from Calbiochem, CA, and Ac-Trp-Glu-His-Asp-aldehyde and
Z-Trp-Glu(O-Me)-His-Asp(O-Me) fluoromethylketone, which can be
obtained from Sigma Aldrich, Germany.
[0337] Exemplary synthetic caspase 6 inhibitors include, for
example, Ac-Val-Glu-Ile-Asp-aldehyde,
Z-Val-Glu-Ile-Asp-fluoromethylketone, and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gl-
u-Ile-Asp-aldehyde (SEQ ID NO: 4), which can be obtained from
Calbiochem. Another exemplary caspase 6 inhibitor includes
Immunocasp-6.
[0338] Exemplary synthetic caspase 7 inhibitors include, for
example, Z-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethylketone,
Ac-Asp-Glu-Val-Asp-aldehyde,
Biotin-Asp-Glu-Val-Asp-fluoromethylketone,
Z-Asp-Glu-Val-Asp-fluoromethylketone,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Gl-
u-Val-Asp-aldehyde (SEQ ID NO: 2), which can be obtained from Sigma
Aldrich, Germany.
[0339] Exemplary synthetic caspase 8 inhibitors include, for
example, Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Ile-Glu-Pro-Asp-aldehyde,
Ac-Ile-Glu-Thr-Asp-aldehyde, Ac-Trp-Glu-His-Asp-aldehyde and
Boc-Ala-Glu-Val-Asp-aldehyde which can be obtained from Bachem
Bioscience Inc., PA. Other exemplary caspase 8 inhibitors include,
for example,
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile-Gl-
u-Thr-Asp-aldehyde (SEQ ID NO: 5) and
Z-Ile-Glu-Thr-Asp-fluoromethylketone, which can be obtained from
Calbiochem, CA.
[0340] Exemplary synthetic caspase 9 inhibitors, include, for
example, Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Leu-Glu-His-Asp-aldehyde,
and Ac-Leu-Glu-His-Asp-chloromethylketone which can be obtained
from Bachem Bioscience Inc., PA. Other exemplary caspase 9
inhibitors include, for example,
Z-Leu-Glu-His-Asp-fluoromethylketone and
Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Gl-
u-His-Asp-aldehyde (SEQ ID NO:6), which can be obtained from
Calbiochem, CA. Another exemplary caspase 9 inhibitor includes
FKBP12/caspase-9 fusion protein.
[0341] The pan-caspase inhibitor may also be an endogenous caspase
inhibitor or a combination of an endogenous caspase inhibitor with
one or more synthetic caspase inhibitors. For example, one useful
class of endogenous caspase inhibitor includes proteins known as
inhibitors of apoptosis proteins (IAPs) (Deveraux et al. (1998)
EMBO J. 17(8): 2215-2223) including bioactive fragments and analogs
thereof. One exemplary IAP includes X-linked inhibitor of apoptosis
protein (XIAP), which has been shown to be a direct and selective
inhibitor of caspase-3, caspase-7 and caspase-9. Another exemplary
IAP includes survivin (see, U.S. Pat. No. 6,245,523;
Papapetropoulos et al. (2000) J. BIOL. CHEM. 275: 9102-9105),
including bioactive fragments and analogs thereof. Survivin has
been reported to inhibit caspase-3 and caspase-7 activity.
[0342] In certain embodiments, the one or more apoptosis inhibitors
may target the inhibitor of apoptosis proteins (IAPs) and second
mitochondria-derived activator of caspases (SMACs). Exemplary
apoptosis inhibitors that target IAPs and SMACs, include, for
example, BIR3 antagonists available from Idun Pharmaceuticals,
capped tripeptide XIAP antagonists from Abbot Laboratories, TWX024,
polyphenylurea derivatives, SMAC-mimetic compounds, embelin, XIAP
antisense and RNAi constructs, AEG35156/GEM.RTM.640 available from
Aegera Therapeutics, HIV-Tat- and polyarginine conjugated SMAC
peptides, and nonpeptide small-molecule SMAC mimetics. It is
contemplated that one or more of the apoptosis inhibitors which
target IAPs and SMACs may be used in combination with one or more
necrostatins (e.g., necrostain-1 and/or necrostatin-4).
[0343] In certain embodiments, the one or more apoptosis inhibitors
may target the TNF-related apoptosis-inducing ligand (TRAIL)
receptors. Exemplary apoptosis inhibitors that target the TRAIL
receptors, include, for example, HGS-ETR1, HGS-ETR2, and HGS-TR2J
available from Human Genome Sciences, and PRO1762 available from
Amgen. It is contemplated that one or more of the apoptosis
inhibitors which target the TRAIL receptors may be used in
combination with one or more necrostatins (e.g., necrostain-1
and/or necrostatin-4).
[0344] In certain embodiments, the one or more apoptosis inhibitors
may target CD95/Fas. Exemplary apoptosis inhibitors that target
CD95/FAS, include, for example, CD95-Fc available from ApoGenix
GmbH. It is contemplated that one or more of the apoptosis
inhibitors which target CD95/Fas may be used in combination with
one or more necrostatins (e.g., necrostain-1 and/or
necrostatin-4).
[0345] In certain embodiments, the one or more apoptosis inhibitors
may be an anti-FasL factors. Exemplary anti-FasL factors include,
for example, anti-FasL neutralizing antibody (available, for
example, from Pharmingen, San Diego, Calif.); peptides and nucleic
acids (for example, anti-FasL aptamers) that bind FasL to prevent
or reduce its binding to its cognate receptor; certain antibodies
and antigen binding fragments thereof and peptides that bind
preferentially to the Fas receptor; antisense nucleotides and
double stranded RNA for RNAi that ultimately reduce or eliminate
the production of either FasL or the Fas receptor; soluble Fas;
soluble FasL; decoy receptor-3 (DcR3) and analogues thereof; matrix
metalloproteinases (MMPs); vasoactive intestinal peptide (VIP);
pituitary adenylate cyclase-activating polypeptide (PACAP);
forskolin; combined use of benazepril and valsartan; nonpeptidic
corticotropin-releasing hormone receptor type 1
(CRH-R.sup.1)-specific antagonists; mimosine; peptides that produce
a defective Fas-FasL complex; platelet-activating factor (PAF); and
endothelin-1 (ET-1). These anti-FasL factors can act as direct or
indirect antagonists of FasL activity.
[0346] In certain embodiments, the one or more apoptosis inhibitors
may target the tumor necrosis factor (TNF). Exemplary apoptosis
inhibitors that target TNF, include, for example, recombinant
TNF-.alpha., adalimumab available from Abbott, infliximab available
from Centocor Ortho Biotech Inc., etanercept from Amgen, CDP571
available from Celltech, and ISIS 104838 (a 2'-O-methoxyethyl
antisense construct against TNF-alpha) available from ISIS
Pharmaceuticals. It is contemplated that one or more of the
apoptosis inhibitors which target TNF may be used in combination
with one or more necrostatins (e.g., necrostain-1 and/or
necrostatin-4).
[0347] In certain embodiments, the one or more apoptosis inhibitors
may target survivin. Exemplary apoptosis inhibitors that target
survivin, include, for example, LY2181308 available from ISIS
Pharmaceuticals and Ad-survivin T34A. It is contemplated that one
or more of the apoptosis inhibitors which target survivin may be
used in combination with one or more necrostatins (e.g.,
necrostain-1 and/or necrostatin-4).
[0348] In certain embodiments, the one or more apoptosis inhibitors
may target the Bcl-2 proteins. Exemplary apoptosis inhibitors that
target the Bcl-2 proteins, include, for example, Bcl-2 blockers
available from Idun Pharmaceuticals and Abbot Laboratories, Gx01
series of compounds available from Gemin X Pharmaceuticals, Bcl-2
small-molecule antagonist, Tetrocarcin-A derivatives available from
Kyowa Hakko Kogyo Co., Chelerythrine, antimycin A derivatives,
HA14-1, synthetic compound binding to the BH3 of Bcl-2, Genasense
available from Sanofi-Aventis, ISIS 22783 available from ISIS
Pharmaceuticals, bispecific Bcl-2/Bcl-XL antisense, BH3 peptides
from Bax, Bak, Bid or Bad, SAHBs, and BH3Is. It is contemplated
that one or more of the apoptosis inhibitors which target the Bcl-2
proteins may be used in combination with one or more necrostatins
(e.g., necrostain-1 and/or necrostatin-4).
[0349] In certain embodiments, the one or more apoptosis inhibitors
may target p53. Exemplary apoptosis inhibitors that target p53,
include, for example, INGN201 available from Invitrogen
Therapeutics, SCH58500 available from Schering-Plough, ONYX-015
available from Onyx Pharmaceuticals, C-terminal p53 peptides, CDB3,
Amifostine, CP31398 available from Pfizer, Prima-1, HPF E6-binding
peptide aptamers, Nutlins available from Roche, Chalcones, Small
peptide compounds, and Pifithrin-.alpha.. It is contemplated that
one or more of the apoptosis inhibitors which target p53 may be
used in combination with one or more necrostatins (e.g.,
necrostain-1 and/or necrostatin-4).
[0350] In certain embodiments, it is contemplated that one or more
necrostatins (e.g., necrostatin-1 and/or necrostatin-4) may be used
in combination with a pan-caspase inhibitor. For example, in one
embodiment, necrostain-1 and/or necrostatin-4 may be used in
combination with Z-VAD available from R&D Systems (Cat. No.
FMK001) and Promega (Cat. No. G7231). In another embodiment,
necrostain-1 and/or necrostatin-4 may be used in combination with
IDN-6556 available from Conatus Pharmaceuticals. In yet another
embodiment, necrostain-1 and/or necrostatin-4 may be used in
combination with IDN-6734 available from Conatus
Pharmaceuticals.
[0351] In certain embodiments, it is contemplated that one or more
necrostatins (e.g., necrostatin-1 and/or necrostatin-4) may be used
in combination with a TNF inhibitor. For example, in one
embodiment, necrostain-1 and/or necrostatin-4 may be used in
combination with adalimumab available from Abbot Laboratories. In
another embodiment, necrostain-1 and/or necrostatin-4 may be used
in combination with etanercept available from Amgen, Inc. In yet
another embodiment, necrostain-1 and/or necrostatin-4 may be used
in combination with infiximab available from Centocor Ortho
Biotech, Inc.
[0352] In certain embodiments, it is contemplated that one or more
necrostatins (e.g., necrostatin-1 and/or necrostatin-4) may be used
in combination with a p53 agonist. For example, in one embodiment,
necrostain-1 and/or necrostatin-4 may be used in combination with
INGN 201 available from Invitrogen Therapeutics. In another
embodiment, necrostain-1 and/or necrostatin-4 may be used in
combination with nutlins, for example, nutlin-3 available from
Cayman Chemical (Cat. No. 10004372). In yet another embodiment,
necrostain-1 and/or necrostatin-4 may be used in combination with
CP31398 available from Tocris Bioscience (Cat. No. 3023).
[0353] In certain embodiments, it is contemplated that one or more
necrostatins (e.g., necrostatin-1 and/or necrostatin-4) may be used
in combination with an anti-FasL factor. For example, in one
embodiment, necrostain-1 and/or necrostatin-4 may be used in
combination with anti-FasL neutralizing antibody available from
Pharmingen (San Diego, Calif.).
[0354] As shown in FIG. 1A and FIG. 19, depending upon the specific
apoptotic inhibitor chosen, it is possible that the apoptotic
inhibitor can modulate both the apoptotic and necrotic pathways,
and depending upon the specific necrosis inhibitor chosen, it is
possible that the necrosis inhibitor can modulate both the necrotic
and apoptotic pathways. For example, a RIP-1 inhibitor may inhibit
both necrotic and apoptotic cell death thus preserving the
viability of the photoreceptor and/or RPE cells in the retina of
the eye of a subject with an ocular condition as disclosed
herein.
[0355] As discussed herein, the methods and compositions of the
invention can preserve the visual function of an eye of a subject
with an ocular condition. Visual function can be measured using one
or more of a variety of methods well-known in the art. For example,
visual function can be assessed by measuring visual acuity. Visual
acuity can be assessed, for example, by using conventional "eye
charts" in which visual acuity is evaluated by the ability to
discern letters of a certain size, with five letters of a given
size present on each line (see, e.g., the "ETDRS" eye chart
described in the Murphy, R. P., CURRENT TECHNIQUES IN OPTHALMIC
LASER SURGERY, 3.sup.rd Ed., edited by L. D. Singerman, and G.
Cascas, Butterworth Heinemann, 2000). Evaluation of visual acuity
may also be achieved by measuring reading speed and reading time.
Visual acuity may be measured to evaluate whether administration of
a necrosis inhibitor and/or an apoptosis inhibitor to the affected
eye preserves or permits improvement of visual acuity (e.g., to
20/40 vision or to 20/20 vision).
[0356] Visual function may also be measured by determining whether
there is an increase in the thickness of the macula (e.g., macula
thickness is 15% thicker than, 35% thicker than, 50% thicker than,
60% thicker than, 70% thicker than, or 80% thicker than a macula
without the treatment as measured by optical coherence tomography
(OCT); an improvement of the photoreceptor cell layer or its
subdivisions as seen in the OCT; an improvement of visual field
(e.g., by at least 10% in the mean standard deviation on the
Humphrey Visual Field Test; an improvement of an electroretinograph
(ERG), a measurement of the electrical response of the retina to
light stimulation, (e.g., to increase ERG amplitude by at least
15%); and or preservation or improvement of multifocal ERG, which
evaluates the response of the retina to multifocal stimulation and
allows characterization of the function of a limited area of the
retina.
[0357] Visual function may also be measured by electrooculography
(EOG), which is a technique for measuring the resting potential of
the retina. EOG is particularly useful for the assessment of RPE
function. EOG may be used to evaluate whether administration of a
necrosis inhibitor and/or an apoptosis inhibitor to the retina of
the affected eye preserves or permits improvement in, for example,
the Arden ratio (e.g., an increase in Arden ratio of at least
10%).
[0358] Visual function may also be assessed through fundus
autofluorescence (AF) imaging, which is a clinical tool that allows
evaluation of the interaction between photoreceptor cells and the
RPE. For example, increased fundus AF or decreased fundus AF has
been shown to occur in AMD and other ocular disorders. Fundus AF
imaging may be used to evaluate whether administration of a
necrosis inhibitor and/or an apoptosis inhibitor to the retina of
the affected eye slows disease progression.
[0359] Visual function may also be assessed by evaluation of
contrast sensitivity, which a measurement of the ability to discern
between luminances of different levels in a static image. An
evaluation of contrast sensitivity may be used to assess whether
administration of a necrosis inhibitor and/or an apoptosis
inhibitor to the retina of the affected eye preserves or permits
improvement in the resolving power of the eye.
[0360] Visual function may also be assessed by microperimetry,
which monitors retinal visual function against retinal thickness or
structure and the condition of the subject's fixation over time.
Microperimetry may be used to assess whether administration of a
necrosis inhibitor and/or an apoptosis inhibitor to the retina of
the affected eye preserves or permits improvement in retinal
sensitivity and fixation.
[0361] It is understood that the methods and compositions described
herein can be used to preserve the viability of photoreceptor and
retinal pigment epithelial cells during treatment of the underlying
conditions, e.g., AMD, RP, etc.
[0362] With regard to preserving the viability of photoreceptor
cells following retinal detachment, it is contemplated herein that
the necrosis inhibitor, e.g., a necrostatin, and/or an apoptosis
inhibitor, e.g., a pan-caspase inhibitor such as Z-VAD or IDN-6556,
may be administered before, during and/or after surgical
reattachment of the detached retina. The necrosis inhibitor and/or
the apoptosis inhibitor may be administered to the mammal from the
time the retinal detachment is detected to the time the retina is
repaired, for example, via surgical reattachment. The retina may be
surgically reattached using procedures known in the art. An
exemplary procedure for surgically reattaching a retina following
detachment includes removing the vitreous fluid and aqueous humor
from the eye and applying a gas, e.g., fluoropropane, to push the
retina against the choroid. Alternatively, buckle surgery, which
does not require removing the vitreous fluid and aqueous humor from
the eye, may be performed to reattach the retina.
[0363] It is understood, however, that under certain circumstances,
it may be beneficial to administer the necrosis inhibitor, e.g., a
necrostatin, and/or the apoptosis inhibitor, e.g., a pan-caspase
inhibitor such as Z-VAD or IDN-6556, even after the retina has been
surgically repaired following retinal detachment. For example, even
after the surgical reattachment of a detached retina in patients
with rhegmatogenous retinal detachments, persistent subretinal
fluid may exist under the fovea as detected by ocular coherence
tomography long after the surgery has been performed (see, Hagimura
et al. (2002) AM. J. OPHTHALMOL. 133:516-520). As a result, even
after surgical repair the retina may still not be completely
reattached to the underlying retinal pigment epithelium and
choroid. Furthermore, when retinal detachments occur secondary to
another disorder, for example, the neovascular form of age-related
macular degeneration and ocular melanomas, as noted above, it may
be beneficial to administer the necrosis inhibitor, the apoptosis
inhibitor, or both the necrosis inhibitor and the apoptosis
inhibitor to the individual while the underlying disorder is being
treated so as to minimize loss of photoreceptor cell viability.
Accordingly, in such cases, it may be beneficial to administer the
necrosis inhibitor and/or the apoptosis inhibitor to the mammal for
one week, two weeks, three weeks, one month, three months, six
months, nine months, one year, two years or more (i) after retinal
detachment has been identified, and/or (ii) after surgical
reattachment of the retina has occurred, and/or (iii) after
detection of an underlying degenerative disorder, so as to minimize
photoreceptor cell death.
[0364] In certain embodiments, the necrosis inhibitor, e.g., a
necrostatin, and/or the apoptosis inhibitor, e.g., a pan-caspase
inhibitor such as Z-VAD or IDN-6556, may be administered locally to
the eye of a subject, e.g., a human subject, following surgical
reattachment of the retina or other treatments of the retina to
preserve or to permit improvement of visual acuity (e.g., to 20/40
vision or to 20/20 vision); to increase the thickness of the macula
(e.g., macula thickness is 15% thicker than, 35% thicker than, 50%
thicker than, 60% thicker than, 70% thicker than, or 80% thicker
than a macula without the treatment as measured by optical
coherence tomography (OCT)); to permit improvement in the
appearance and structure of the photoreceptor cell layer and its
supporting RPE, to permit the improvement of visual field (e.g., by
at least 10% in the mean standard deviation on the Humphrey Visual
Field Test; and/or to permit the improvement of an
electroretinograph (ERG), a measurement of the electrical response
of the retina to light stimulation, (e.g., to increase ERG
amplitude by at least 15%).
[0365] In certain embodiments, the necrosis inhibitor, e.g., a
necrostatin, and/or the apoptosis inhibitor, e.g., a pan-caspase
inhibitor such as Z-VAD or IDN-6556, are administered locally to
the eye of a mammal by intravitreal injection. Both agents may be
administered on the day of diagnosis and/or the same day that the
retina is surgically reattached or has gone through other
treatments and/or in the post-operative period. The agents may then
be administered every three days, every five days, or every seven
days until the mammal, e.g., a human, has improved vision (e.g.,
visual acuity has improved to 20/40 vision or to 20/20 vision), the
thickness of the macula has increased (e.g., macula thickness is
15% thicker than, 35% thicker than, 50% thicker than, 60% thicker
than, 70% thicker than, or 80% thicker than without treatment as
measured by OCT); the appearance of the photoreceptor cell layer
and RPE as detected by OCT; the visual field has improved by at
least 10% in the mean standard deviation as determined by Humphrey
Visual Field testing; and/or the mammal's retina shows an increased
response to light stimulation (e.g., at least a 15% increase in
amplitude as determined by electroretinography).
[0366] The necrosis inhibitor and the apoptosis inhibitor can be
administered by the same route or by different routes. The necrosis
inhibitor and/or the apoptosis inhibitor may be administered
locally to the eye, for example, by intravitreal, intraocular,
intraorbital, subconjuctival, subretinal or transscleral routes.
For example, the necrosis inhibitor and/or the apoptosis inhibitor
may be administered locally to the eye by intravitreal injection.
It is contemplated that local modes of administration may reduce or
eliminate the incidence of potential side effects (e.g., systemic
toxicity) that may occur during systemic administration.
[0367] Alternatively, the necrosis inhibitor and/or the apoptosis
inhibitor may be administered systemically, e.g., by oral or
parenteral routes. Parenteral routes include, for example,
intravenous, intrarterial, intramuscular, intradermal,
subcutaneous, intranasal and intraperitoneal routes.
[0368] The necrosis inhibitor and the apoptosis inhibitor may be
administered to a subject simultaneously or sequentially. It will
be appreciated that when administered simultaneously, the necrosis
inhibitor and the apoptosis inhibitor may be in the same
pharmaceutically acceptable carrier (e.g., solubilized in the same
viscoelastic carrier that is introduced into the eye) or the two
drugs may be dissolved or dispersed in separate pharmaceutical
carriers, which are administered at the same time. Alternatively,
the drugs may be provided in separate dosage forms and administered
sequentially. For example, in some embodiments, the necrostatin may
be administered before the pan-caspase inhibitor. In other
examples, the pan-caspase inhibitor may be administered before the
necrostatin. In addition, it is appreciated that, in some
embodiments, a single active agent may inhibit both necrosis and
apoptosis.
[0369] Administration may be provided as a periodic bolus (for
example, intravitreally or intravenously) or as continuous infusion
from an internal reservoir (for example, from an implant disposed
at an intra- or extra-ocular location (see, U.S. Pat. Nos.
5,443,505 and 5,766,242)) or from an external reservoir (for
example, from an intravenous bag, or a contact lens slow release
formulation system). The necrosis inhibitor and/or the apoptosis
inhibitor may be administered locally, for example, by continuous
release from a sustained release drug delivery device immobilized
to an inner wall of the eye or via targeted transscleral controlled
release into the choroid (see, for example, PCT/US00/00207,
PCT/US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS.
SCI.41:1181-1185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS.
SCI.41:1186-1191). A variety of devices suitable for administering
the disclosed necrosis and/or apoptosis inhibitors locally to the
inside of the eye are known in the art. See, for example, U.S. Pat.
Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and 6,375,972, and
PCT/US00/28187.
[0370] The necrosis inhibitor and/or the apoptosis inhibitor may be
solubilized in a carrier, for example, a viscoelastic carrier, that
is introduced locally into the eye. One or both inhibitors also may
be administered in a pharmaceutically acceptable carrier or vehicle
so that administration does not otherwise adversely affect the
recipient's electrolyte and/or volume balance. The carrier may
comprise, for example, physiologic saline or other buffer system.
In exemplary embodiments, the necrostatin, the pan-caspase
inhibitor, or both the necrostatin and the pan-caspase inhibitor
may be solubilized in PBS or another aqueous buffer by sonication.
Alternatively, one or both drugs may be solubilized using
conventional solvent or solubilization systems, for example,
dimethyl sulfoxide (DMSO), dimethoxyethane (DME), dimethylformamide
(DMF), cyclodextran, micelles, liposomes, liposomal agents, and
other solvents known in the art to aid in the solubilization and
administration of hydrophobic agents.
[0371] In other embodiments, the necrosis inhibitor and/or the
apoptosis inhibitor may be solubilized in a liposome or
microsphere. Methods for delivery of a drug or combination of drugs
in liposomes and/or microspheres are well-known in the art.
[0372] In addition, it is contemplated that the necrosis inhibitor
and/or the apoptosis inhibitor may be formulated so as to permit
release of one or both inhibitors over a prolonged period of time.
A release system can include a matrix of a biodegradable material
or a material, which releases the incorporated active agents. The
active agents can be homogeneously or heterogeneously distributed
within a release system. A variety of release systems may be useful
in the practice of the invention, however, the choice of the
appropriate system will depend upon the rate of release required by
a particular drug regime. Both non-degradable and degradable
release systems can be used. Suitable release systems include
polymers and polymeric matrices, non-polymeric matrices, or
inorganic and organic excipients and diluents such as, but not
limited to, calcium carbonate and sugar (for example, trehalose).
Release systems may be natural or synthetic. However, under certain
circumstances, synthetic release systems are preferred because
generally they are more reliable, more reproducible and produce
more defined release profiles. The release system material can be
selected so that inhibitors having different molecular weights are
released by diffusion through or degradation of the material.
[0373] Representative synthetic, biodegradable polymers include,
for example: polyamides such as poly(amino acids) and
poly(peptides); polyesters such as poly(lactic acid), poly(glycolic
acid), poly(lactic-co-glycolic acid), and poly(caprolactone);
poly(anhydrides); polyorthoesters; polycarbonates; and chemical
derivatives thereof (substitutions, additions of chemical groups,
for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof. Representative synthetic,
non-degradable polymers include, for example: polyethers such as
poly(ethylene oxide), poly(ethylene glycol), and
poly(tetramethylene oxide); vinyl polymers-polyacrylates and
polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl
methacrylate, acrylic and methacrylic acids, and others such as
poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl
acetate); poly(urethanes); cellulose and its derivatives such as
alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various
cellulose acetates; polysiloxanes; and any chemical derivatives
thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art),
copolymers and mixtures thereof.
[0374] One of the primary vehicles currently being developed for
the delivery of ocular pharmacological agents is the
poly(lactide-co-glycolide) microsphere for intraocular injection.
The microspheres are composed of a polymer of lactic acid and
glycolic acid, which are structured to form hollow spheres. These
spheres can be approximately 15-30 .mu.m in diameter and can be
loaded with a variety of compounds varying in size from simple
molecules to high molecular weight proteins such as antibodies. The
biocompatibility of these microspheres is well established (see,
Sintzel et al. (1996) EUR. J. PHARM. BIOPHARM. 42: 358-372), and
microspheres have been used to deliver a wide variety of
pharmacological agents in numerous biological systems. After
injection, poly(lactide-co-glycolide) microspheres are hydrolyzed
by the surrounding tissues, which cause the release of the contents
of the microspheres (Zhu et al. (2000) NAT. BIOTECH. 18: 52-57). As
will be appreciated, the in vivo half-life of a microsphere can be
adjusted depending on the specific needs of the system.
[0375] Throughout the description, where compositions are described
as having, including, or comprising specific components, or where
processes are described as having, including, or comprising
specific process steps, it is contemplated that compositions of the
present invention also consist essentially of, or consist of, the
recited components, and that the processes of the present invention
also consist essentially of, or consist of, the recited processing
steps. Further, it should be understood that the order of steps or
order for performing certain actions are immaterial so long as the
invention remains operable. Moreover, two or more steps or actions
may be conducted simultaneously.
EXAMPLES
[0376] The invention is further illustrated by the following
examples, which are provided for illustrative purposes only, and
should not be construed as limiting the scope or content of the
invention in any way.
[0377] In the examples described herein, all animal experiments
adhered to the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Vision Research,
and protocols were approved by the Animal Care Committee of the
Massachusetts Eye and Ear Infirmary. Adult male Brown Norway rats
(200 to 300 g) and WT C57BL/6 mice were purchased from Charles
River Laboratories (Wilmington, Mass.). The mice were fed standard
laboratory chow and allowed free access to water in an
air-conditioned room with a 12 hour light/12 hour dark cycle. RIP-3
knockout (RIP-3-/-) mice were kindly provided by Dr. V. M. Dixit
(Genentech, San Francisco, Calif.). RIP-3-/- mice were generated as
described previously and backcrossed to C57BL/6 mice (Newton et al.
(2004) MOL CELL BIOL 24:1464-9). Rd10 mice were purchased from the
Jackson Laboratory (Bar Harbor, Me.). CCL2/Cx3Cr1 double knockout
mice were kindly provided by Dr. Chi-Chao Chan (National Eye
Institute and National Institutes of Health, Bethesda, Md.).
CCL2/Cx3Cr1/RIP-3 triple knockout mice were generated by breeding
CCL2/Cx3Cr1 double knockout mice with RIP-3 knockout mice.
[0378] The following reagents are utilized: Z-VAD (Alexis, Plymouth
Meeting Pa.), IDN-6556 (kindly provided by TetraLogics
Pharmaceuticals), a Nec-1 compound of Formula I-C(a kind gift from
Dr. J. Yuan, Harvard Medical School, Boston, Mass.), and a Nec-4
compound of Formula IV-A (kindly provided by TetraLogics
Pharmaceuticals).
[0379] Experimental retinal detachment was induced as previously
described (Hisatomi T et al. (2001) AM. J. PATHOL. 158:1271-1278.
Briefly, a 30-gauge needle was inserted into the subretinal space
via an external transscleral transchoroidal approach, and 1% sodium
hyaluronate (Provisc; Alcon, Fort Worth, Tex.) containing either
vehicle (control), Z-VAD (300 .mu.M), IDN-6556 (200 .mu.M), a Nec-1
compound of Formula I-C(400 .mu.M), and/or Nec-4 compound of
Formula IV-A (200 .mu.M) was gently injected into the subretinal
space to enlarge the retinal detachment.
[0380] All values disclosed below were expressed as the mean.+-.SD.
Statistical differences between two groups were analyzed by
Mann-Whitney U test. Multiple group comparison was performed by
ANOVA followed by Tukey-Kramer adjustments. Differences were
considered significant at P<0.05.
Example 1: Increased Expression of RIP-3 and RIP-1 after Retinal
Detachment
[0381] Photoreceptor death after retinal detachment has been
thought to be caused mainly by apoptosis (Cook et al. (1995) INVEST
OPHTHALMOL VIS SCI 36:990-996; Arroyo et al. (2005) AM J OPHTHALMOL
139:605-610. Although caspase-dependent pathway is known to be
activated after retinal detachment, caspase inhibition by
pan-caspase inhibitor fails to prevent photoreceptor death (Zacks
et al. (2003) INVEST OPHTHALMOL VIS SCI 44:1262-1267; Hisatomi et
al. (2001) AM J OPHTHALMOL 158:1271-1278). In this example, other
pathways leading to photoreceptor death were investigated. In
particular, the expression levels of RIP-1 and RIP-3 proteins were
measured after retinal detachment. RIP-3 is a key regulator of
RIP-1 kinase activation (Galluzzi L et al. (2009) J. MOL. CELL
BIOL.) and its expression level has been shown to correlate with
responsiveness to programmed necrosis (He S et al. (2009) CELL
137:1100-1111). As described below, it was demonstrated that
necrotic photoreceptor death occurs after retinal detachment
although its frequency is about half that of apoptosis.
[0382] Total RNA extraction and reverse transcription were
performed as previously reported (Nakazawa et al. (2006) MOL. VIS.
12:867-878; Hisatomi et al. (2008). J. CLIN. INVEST.
118:2025-2038). A real-time PCR assay was performed with Prism 7700
Sequence Detection System (Applied Biosystems, Foster City,
Calif.). TaqMan Gene Expression assays were used to check the
expression of RIP-1 (Rn01757378_m1) and RIP-3 (Rn00595154_m1). For
relative comparison of each gene, the Ct value of real-time PCR
data was analyzed with the AACt method and normalized by an
endogenous control (18S ribosomal RNA).
[0383] Partial sequence of mouse RIP-3 gene was amplified by RT-PCR
using primers AGCACAGGACACATCAGTTGG and CTTGAGGCAGTAGTTCTTGGTG, and
cloned into the pCR-II vector (Invitrogen). Digoxigenin-labeled
riboprobe was hybridized at 61.degree. C. overnight, followed by
stringent washes. The cryosections were then treated with an
alkaline phosphatase-conjugated antidigoxigenin antibody (Roche,
Indianapolis, Ind.). Hybridization signals were visualized with BM
purple AP substrate (Roche).
[0384] The vitreous and neural retina combined was collected on day
3 after retinal detachment. Samples were run on 4-12%
SDS-polyacrylamide gel electrophoresis and transferred onto PVDF
membrane. After blocking with 3% nonfat dried milk, the membrane
was reacted with RIP-3 (1:10000; Sigma, Saint Louis, Mich.), RIP-1
(1:2000; BD Biosciences), phosphoserine (1:2000; Enzo, Plymouth
Meeting, Pa.) or anti-phosphorylated NF-icB p.sup.65 (1:1000; Cell
signaling) antibody. Membranes were then developed with enhanced
chemiluminescence. Lane-loading differences were normalized by
(3-tubulin (1:1000; Cell Signaling).
[0385] Equal amount of retinal lysates (1 mg) were incubated with 1
.mu.g anti-RIP-1 antibody (BD Biosciences) and 20 .mu.l of Protein
A/G Agarose beads (Thermo Scientific, Rockford, Ill.) at 4.degree.
C. overnight. Beads were washed 5 times with lysis buffer and TBS,
and the immunopellets were then subjected to Western blotting.
[0386] The expression levels of RIP-3 and RIP-1 mRNA were assessed
in the retina three days after retinal detachment by quantitative
real-time PCR. This time point was chosen because photoreceptor
death peaks at three days after retinal detachment (Hisatomi T et
al. (2001) AM. J. PATHOL. 158:1271-1278). FIG. 1B-C provide graphs
showing the quantitative real-time PCR analysis for RIP-3 (FIG. 1B)
and RIP-1 (FIG. 1C) in control retina without retinal detachment
and in retina three days after retinal detachment (n=9 each; **,
P<0.01). RIP-3 expression increased 12-fold after retinal
detachment compared to that in untreated retina (P=0.0003; FIG.
1B). RIP-1 expression also increased 2-fold after retinal
detachment (P=0.0031; FIG. 1C).
[0387] Protein expression of RIP-3 and RIP-1 after retinal
detachment (n=4 each) was analyzed by Western blot. Lane-loading
differences were normalized by levels of .beta.-tubulin. For RIP-3
analysis, spleen samples from wild-type (WT) and RIP-3-/- animals
were used as positive and negative controls, respectively. The bar
graphs indicate the relative level of RIP-3 and RIP-1 to
.beta.-tubulin by densitometric analysis, reflecting the results
from four independent experiments (*, P<0.05). Western blot
analysis confirmed that RIP-3 protein expression increased over
10-fold after retinal detachment (P=0.0209; FIG. 1D, black
arrowhead: RIP-3; white arrowhead: non-specific band).
[0388] In situ hybridization analysis of RIP-3 was also conducted.
RIP-3 signal was detected in retinal tissues, especially in the
outer nuclear layer (ONL) after retinal detachment. The retina from
RIP-3-/- animals was used as negative control. In situ
hybridization showed that RIP-3 signal was detected in neurosensory
retina, especially in the ONL, after retinal detachment (FIG. 2;
GCL: ganglion cell layer; INL: inner nuclear layer; scale bar, 50
am).
[0389] These data indicate that RIP-mediated programmed necrosis is
involved in photoreceptor loss after retinal detachment.
Example 2: Necrosis Inhibitor Prevents Retinal Detachment-Induced
Photoreceptor Death in Combination with a Caspase Inhibitor
[0390] Necrostatin-1 (Nec-1) is a potent and selective inhibitor of
programmed necrosis, which targets RIP-1 kinase activity (Degterev
A et al. (2008) NAT. CHEM. BIOL. 4:313-321). The effectiveness of a
RIP-1 kinase inhibitor in combination with a capsase inhibitor to
prevent retinal detachment induced photoreceptor death was
investigated.
[0391] RIP-1 is phosphorylated at several serine residues in its
kinase domain during programmed necrosis and Nec-1 inhibits this
phosphorylation (Degterev (2008) supra; Cho Y et al. (2009) Cell
137:1100-1111). To investigate the role of RIP-1 kinase in retinal
detachment-induced photoreceptor death, the phosphorylation status
of RIP-1 was first assessed by immunoprecipitating RIP-1 from the
retina (FIG. 3A) and then blotting with an anti-phosphoserine
antibody. RIP-1 was immunoprecipitated from retinal lysates. One mg
of retinal lysates were incubated with anti-RIP-1 antibody and
Protein A/G agarose beads. Extracts before (Pre) and after (Post)
immunoprecipitation and the immunopellets (IP) were run on a 4-12%
SDS-PAGE and blotted with anti-RIP-1 antibody. Anti-RIP-1 antibody
alone was used as negative control. Extracts
post-immunoprecipitation showed almost complete immunodepletion of
RIP-1 from the retinal extract.
[0392] To determine the effect of Nec-1 and a pan-caspase inhibitor
on the phosphorylation of RIP-1, Z-VAD (300 rM) and/or a Nec-1
compound of Formula I-C(400 .mu.M) were injected subretinally in
the animal at the time of retinal detachment induction. The dosage
of compounds was selected based on previous studies which
established that the half-life of the compounds is around 6 hours
in the subretinal space. RIP-1 was immunoprecipitated from lysates
of untreated retina and of retina treated with either vehicle,
Nec-1, Z-VAD, or Nec-1+Z-VAD at two days after retinal detachment.
Phosphorylation of RIP-1 was then assessed by Western blot
analysis. After retinal detachment, RIP-1 phosphorylation was
elevated in Z-VAD-treated retina compared to untreated retina (FIG.
3B). As shown in FIG. 3B, Nec-1 and Z-VAD combination treatment
substantially inhibited this increase in RIP-1 phosphorylation.
[0393] Photoreceptor death was also assessed by TUNEL analysis at
three days after retinal detachment in retina treated with either
vehicle, Nec-1, Z-VAD or Nec-1+Z-VAD. TUNEL and quantification of
TUNEL positive cells in ONL were performed as previously described
(Nakazawa T et al. (2007) PROC. NATL. ACAD. SCI. USA 104:2425-2430)
by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit
(Millipore, Billerica, Mass.). The ratio of the thickness of the
ONL to the thickness of neuroretina in the central area of the
detached retina was determined by National Institutes of Health
ImageJ software and standardized by that in the attached retina.
The data are expressed as normalized ONL thickness ratio
[(ONL/neuroretina thickness in detached retina)/(ONL/neuroretina
thickness in attached retina)]. Nine sections for each eye specimen
were randomly selected, and the central area of detached retina and
the mid-peripheral region of attached retina were photographed. The
ONL thickness ratio was measured at ten points in each section by
masked observers. The data are expressed as normalized ONL
thickness ratio [(ONL/neuroretina thickness in detached
retina/(ONL/neuroretina thickness in attached retina)]. FIG. 4A
shows TUNEL (green) and DAPI (blue) staining in detached retina
treated with vehicle, Z-VAD, Nec-1 (a Nec-1 of Formula I-C) or
Nec-1+Z-VAD on day three after retinal detachment. FIG. 4B-C show
quantification of TUNEL-positive photoreceptors (FIG. 4B) and ONL
thickness ratio (FIG. 4C) on day three (vehicle, n=12; Z-VAD, n=12;
Nec-1, n=6; Z-VAD+Nec-1, n=12) and day five (vehicle, n=8; Z-VAD,
n=8; Nec-1, n=6; Z-VAD+Nec-1, n=8) after retinal detachment. (GCL:
ganglion cell layer, INL: inner nuclear layer. *, P<0.05. Scale
bar, 100 m).
[0394] Treatment with Z-VAD or Nec-1 alone showed no significant
effect on the number of TUNEL-positive cells in the ONL
(1134.7.+-.297.5 and 1067.5.+-.95.5 cells/mm.sup.2, respectively),
as compared with vehicle treatment (1366.3.+-.103.7 cells/mm.sup.2;
FIG. 4A-B). In comparison, combined treatment with Nec-1+Z-VAD
significantly reduced the number of TUNEL-positive cells in the ONL
(573.1.+-.154.3 cells/mm.sup.2, P<0.05; FIG. 4A-B). The
appearance of TUNEL-positive cells was decreased to approximately
200 cells/mm.sup.2 in each group five days after retinal detachment
(FIG. 4B). Although detection of DNA fragmentation by TUNEL is used
as a marker of apoptosis, it has been reported that necrosis,
programmed or otherwise, also yields DNA fragments that react with
TUNEL in vivo, rendering it difficult to discriminate between
apoptosis and necrosis (Grasl-Kraupp B et al. (1995) HEPATOLOGY
21:1465-1468).
[0395] The thickness ratio of ONL to the total retinal thickness in
detached retina compared to normal attached retina was also
measured. The normalized ONL thickness ratio was reduced to
0.65.+-.0.10 in vehicle-treated detached retina three days (bars
with stippled shading in FIG. 4C) after retinal detachment.
Treatment with Z-VAD or Nec-1 alone had no protective effect on the
reduction of ONL thickness ratio (0.72.+-.0.08 and 0.71.+-.0.09,
respectively); whereas Z-VAD+Nec-1 treatment significantly
prevented the reduction in retinal thickness (0.92.+-.0.06,
P<0.05; FIGS. 4A and 4C). Photoreceptor loss was more severe
five days (bars with solid shading in FIG. 4C) after retinal
detachment; with a normalized ONL thickness ratio reduced to
0.52.+-.0.08 in vehicle-treated animals, 0.54.+-.0.10 in
Z-VAD-treated animals and 0.58.+-.0.12 in Nec-1-treated animals,
with substantial, synergistic preservation of retinal thickness in
animals treated with Nec-1+Z-VAD (0.81.+-.0.06, P<0.05; FIG.
4C).
[0396] Treatment with both Nec-1 and Z-VAD also showed efficient
neuroprotection when the compounds were injected intravitreally one
day after retinal detachment induction. FIG. 5A-B shows
quantification of TUNEL-positive photoreceptors (FIG. 5A) and ONL
thickness ratio (FIG. 5B) on day three after retinal detachment in
rats (n=4-5). Five .mu.l of Nec-1 (2 mM) and/or Z-VAD (3 mM) were
injected intravitreally one day after retinal detachment-induced
photoreceptor death. Treatment with Nec-1+Z-VAD significantly
decreased the number of TUNEL-positive cells and prevented the
reduction of ONL thickness ratio after retinal detachment. *,
P<0.05; ** P<0.01.
[0397] The effects of another necrostatin, Nec-4 (Teng et al.,
(2008) BIOORG MED CHEM LETT, 18: 3219-3223) and/or another
pan-caspase inhibitor (IDN-6556-labeled PCI in FIG. 6A-F) (Hoglen N
C et al. (2004) J PHARMACOL EXP THER 309:634-640) on retinal
detachment-induced photoreceptor death were also assessed.
TUNEL-positive photoreceptors were quantitated (FIG. 6 A, 6C, 6E)
and ONL thickness ratio was measured (FIGS. 6 B, 6D, and 6F) on day
three after retinal detachment in rats (n=5-6). Nec-4 (a compound
of Formula IV-A), IDN-6556 and/or Z-VAD were injected subretinally
at the doses indicated in FIG. 6A-F. Nec-4 or IDN-6556 alone did
not show any neuroprotective effect (FIG. 6 A-D), while
Nec-4+IDN-6556 and Nec-4+Z-VAD combination treatments significantly
suppressed photoreceptor loss after retinal detachment (FIG. 6E-F)
(P<0.05; **, P<0.01). Nec-4 and IDN-6556 were prepared as
described in Teng et al., (2008) BIOORG MED CHEM LETT, 18:
3219-3223 and Hoglen et al. ((2004) J PHARMACOL EXP THER
309:634-640), respectively. These data indicate that RIP-1 kinase
mediates photoreceptor death after retinal detachment in the
presence of a pan-caspase inhibitor. As a result, simultaneous
inhibition of both caspase and RIP-1 kinase pathways significantly
limits photoreceptor death after retinal detachment. As
demonstrated herein, the co-administration of a RIP-1 inhibitor,
e.g., necrostatin-1 or necrostatin-4, and a pan-caspase inhibitor,
e.g., Z-VAD or IDN-6556, achieved a synergistic effect compared to
treatment with either drug alone. Co-administration of a RIP-1
inhibitor, e.g., necrostatin-1 or necrostatin-4, and a pan-caspase
inhibitor, e.g., Z-VAD or IDN-6556, may be used to prevent vision
loss in various retinal disorders associated with retinal
detachment and other degenerative diseases.
[0398] Consistent with our findings, Arimura et al. showed that the
vitreous level of high-mobility group box i protein, which is known
to be released from necrotic cells but not apoptotic cells, is
increased in human eyes with retinal detachment (Arimura et al.
(2009) LAB INVEST 89:278-289; Scaffidi et al. (2002) Nature
418:191-195). Furthermore, we found that Z-VAD treatment decreases
apoptosis but increases necrotic photoreceptor death after retinal
detachment, and that Nec-1 co-treatment effectively suppresses
necrotic photoreceptor death. These findings clearly demonstrate
programmed necrosis as an essential and complementary mechanism of
photoreceptor death, which is further revealed when caspases are
inhibited (FIG. 15A-C). In other retinal degenerative conditions
such as inherited retinal degeneration and light-induced retinal
injury, it has been shown that the photoreceptor death is not
prevented by Z-VAD (Donovan & Cotter (2002) CELL DEATH DIFFER
9:1220-1231; Sanges et al. (2006) PROC NATL ACAD SCI USA
103:17366-17371; Murakami et al. (2008) AM J PATHOL 173-1326-1338),
and thus it may be possible that programmed necrosis may underlie
the death execution in these diseases as well.
Example 3: Caspase Inhibition Shifts Retinal Detachment-Induced
Photoreceptor Death from Apoptosis to Programmed Necrosis
[0399] The morphology of photoreceptors after retinal detachment
was examined by transmission electron microscopy (TEM) and
propidium iodide (PI) staining and the effect of treatment with
Z-VAD and/or a Nec-1 compound of Formula I-C was analyzed.
[0400] TEM was performed as previously described (Hisatomi T et al.
(2008). J. CLIN. INVEST. 118:2025-2038). The specimens were
observed with Philips CM10 electron microscope. Over 200
photoreceptors each eye were photographed and subjected to
quantification of cell death modes in a masked fashion.
Photoreceptors showing cellular shrinkage and nuclear condensation
were defined as apoptotic cells, while photoreceptors associated
with cellular and organelle swelling and discontinuities in nuclear
and plasma membrane were defined as necrotic cells. The presence of
electron-dense granular materials were reported to occur subsequent
to both apoptotic and necrotic cell death and cells with these
findings were labeled simply as end-stage cell death/unclassified
(Hisatomi T et al. (2003) AM. J. PATHOL. 162:1869-1879; Erickson P
A et al. (1983) INVEST. OPHTHALMOL. VIS. SCI. 24:927-942).
[0401] FIG. 7A-E show TEM photomicrographs in the ONL on day three
after retinal detachment in retina treated with vehicle (FIG. 7A),
Z-VAD (FIG. 7B) or Nec-1+Z-VAD (the Nec-1 compound was a compound
of Formula I-C) (FIG. 7C). On day three after retinal detachment,
photoreceptor death caused by apoptosis was almost twice that
caused by necrosis in vehicle-treated retina (% apoptotic cells:
21.7.+-.1.3%, necrotic cells: 13.3.+-.1.0%, unclassified:
4.4.+-.1.4%; FIGS. 7A and 7E). Nec-1 treatment did not affect the
percentage of apoptotic and necrotic cells after retinal detachment
(% apoptotic cells: 19.8.+-.2.0%, necrotic cells: 13.1.+-.1.4%,
unclassified: 2.8.+-.0.4%; FIGS. 7 B and 7E). In comparison, Z-VAD
treatment significantly decreased apoptotic photoreceptor death and
increased necrotic cell death (% apoptotic cells: 11.4.+-.1.2%,
necrotic cells: 21.9.+-.2.3%, unclassified: 5.6.+-.1.4%, P<0.05;
FIGS. 7 C and 7E). However, Nec-1 combined with Z-VAD substantially
prevented the switch to necrotic cell death and led to a decrease
in both forms of cell loss (% apoptotic cells: 6.9.+-.2.9%,
necrotic cells: 9.8.+-.0.7%, unclassified: 1.0.+-.0.2%, P<0.01;
FIGS. 7D and 7E). FIG. 7E shows the quantification of apoptotic and
necrotic photoreceptor death after retinal detachment (n=4
each).
[0402] These results indicate that Z-VAD, as compared with vehicle,
increased necrotic cells and decreased apoptotic cells (P<0.05),
and that Nec-1 combined with Z-VAD significantly suppressed
necrotic cell death (P<0.01, vs. Z-VAD) (scale bar, 50
.mu.m).
[0403] In vivo propidium iodide (PI) staining was performed by
injecting 5 .mu.l of PI (50 .mu.g/ml) into the subretinal space
three days after retinal detachment. After two hours, the eyes were
enucleated and 10 .mu.m thick cryosections were cut, air-dried, and
fixed in 100% ethanol. DAPI was used to counterstain the nuclei.
The center of the detached retina was photographed by fluorescence
microscope, and the number of PI-positive cells in ONL was analyzed
by ImageJ software.
[0404] FIG. 8A-D shows PI staining (FIG. 8A) and quantification of
PI-positive photoreceptors (B) on day three after retinal
detachment in retina treated with vehicle, Z-VAD or Nec-1+Z-VAD
(the Nec-1 compound was a compound of Formula I-C) in wild-type
retina (n=6 each). Consistent with the transmission electron
microscopy findings, subretinal injection of PI prior to
enucleation, a technique to demonstrate cells with disrupted cell
membrane, showed increased number of PI-positive photoreceptors in
Z-VAD-treated retina (817.4.+-.104.2 cells/mm.sup.2)compared to
vehicle-treated retina three days after retinal detachment
(366.5.+-.82.1 cells/mm.sup.2, P<0.05; FIG. 8A-B). Treatment
with Nec-1+Z-VAD significantly suppressed the number of PI-positive
cells in the ONL (16.0.+-.7.6 cells/mm.sup.2, P<0.05: FIG.
8A-B). (*, P<0.05; **, P<0.01; scale bar, 100 .mu.m.)
[0405] Collectively, these results indicate that programmed
necrosis as well as apoptosis are involved in photoreceptor death
after retinal detachment, and that RIP-1-mediated programmed
necrosis becomes a predominant form of photoreceptor death when the
caspase-dependent apoptotic pathway is inhibited.
Example 4: RIP-3 Deficiency Inhibits Induction of Programmed
Necrosis and Prevents Photoreceptor Death After Retinal
Detachment
[0406] The role of RIP-3 in RIP-mediated programmed necrosis in
photoreceptor loss after retinal detachment was investigated in
RIP-3 knockout (RIP-3-/-) mice. TUNEL assays were used to measure
the amount of apoptotic and necrotic cell death (Grasl-Kraupp et
al., HEPTALOGY (1995) 21(5):1465-8). TUNEL and quantification of
TUNEL positive cells in ONL were performed as described above by
using the ApopTag Fluorescein In Situ Apoptosis Detection Kit
(Millipore, Billerica, Mass.). The ratio of the ONL thickness to
the thickness of neuroretina in the central area of the detached
retina was determined by National Institutes of Health ImageJ
software and standardized by that in the attached retina. The data
are expressed as normalized ONL thickness ratio [(ONL/neuroretina
thickness in detached retina)/(ONL/neuroretina thickness in
attached retina)].
[0407] FIG. 9A shows TUNEL (green) and DAPI (blue) staining in
detached retina in WT and RIP-3-/- mice treated with vehicle,
Z-VAD, Nec-1, or Nec-1+Z-VAD (the Nec-1 compound was a compound of
Formula I-C) at three days after retinal detachment. FIG. 9B shows
quantification of TUNEL-positive photoreceptors on day three after
retinal detachment (vehicle in WT mice, n=6; vehicle in RIP-3-/-
mice, n=7; Z-VAD in RIP-3-/- mice, n=7; Nec-1+Z-VAD in RIP-3-/-
mice, n=6). FIG. 9C shows ONL thickness on day three (vehicle in WT
mice, n=6; vehicle in RIP-3-/- mice, n=7; Nec-1 in RIP-3-/- mice,
n=8; Z-VAD in RIP-3-/- mice, n=7; Nec-1+Z-VAD in RIP-3-/- mice,
n=6) and day five (vehicle in WT mice, n=7; vehicle in RIP-3-/-
mice, n=6; Nec-1 in RIP-3-/- mice, n=6; Z-VAD in RIP-3-/- mice,
n=6; Nec-1+Z-VAD in RIP-3-/- mice, n=6) after retinal detachment
(*, P<0.05; **, P<0.01. Scale bar, 50 m).
[0408] Without retinal detachment, the morphology of the retina and
ONL thickness ratio were similar in RIP-3-/- and wild-type (WT)
mice. Three days after retinal detachment, RIP-3-/- mice showed
significantly less TUNEL-positive cells (804.7.+-.204.5
cells/mm.sup.2) than WT mice (1668.7.+-.305.8 cells/mm.sup.2,
P<0.01; FIGS. 9A and 9B). In contrast to WT animals, Z-VAD
treatment in RIP-3-/- mice further decreased the number of
TUNEL-positive cells after retinal detachment (407.7.+-.188.9
cells/mm.sup.2, P<0.05, vs. RIP-3-/- mice treated with vehicle),
whereas Nec-1 did not provide any additional effect (786.7.+-.278.7
cells/mm.sup.2; FIGS. 9A and 9B). RIP-3-/- retinas exhibited
preserved ONL thickness ratio, which was augmented by Z-VAD
treatment, on day 3 and 5 after retinal detachment (FIG. 9C).
[0409] In summary, suppression of RIP-3 gene expression in the
RIP-3-/- mice significantly reduced the number of TUNEL positive
cells consistent with RIP-3 playing a role in mediating programmed
necrosis. Treatment with Z-VAD in the RIP-3-/- mice further reduced
the number of TUNEL-positive cells in the ONL. In comparison,
treatment with Z-VAD and Nec-1 did not result in further reductions
in the number of TUNEL-positive cells in the ONL in the RIP-3-/-
mice three days after retinal detachment (FIG. 9B) suggesting that
the amount of necrotic cell death in RIP-3-/- mice is similar to
the amount of necrotic cell death observed in mice treated with
Nec-1.
[0410] The thickness ratio of ONL to the total retinal thickness in
detached retina compared to normal attached retina was also
measured (FIG. 9C). The normalized ONL thickness ratio increased in
RIP-3-/- mice compared to WT mice five days after retinal
detachment (WT: 0.644.+-.0.064; RIP-3-/-: 0.754.+-.0.049)
suggesting that RIP-3-/- mice have a protective effect compared to
WT mice following retinal detachment. Treatment with Nec-1 in
RIP-3-/- mice did not provide any further protection following
retinal detachment (RIP-3-/-+Nec1: 0.761.+-.0.049); whereas
treatment with Z-VAD in RIP-3-/- mice significantly prevented a
reduction in retinal thickness (RIP-3-/-+ZVAD: 0.841.+-.0.042).
[0411] The morphology of the RIP-3-/- retina after retinal
detachment was also assessed by TEM. In WT mice, the percentage of
necrotic photoreceptors was significantly increased by Z-VAD
treatment (% apoptotic cells: 13.3.+-.1.5%, necrotic cells:
22.1.+-.1.0%, unclassified: 1.0.+-.0.6%) compared to vehicle
treatment (% apoptotic cells: 22.2.+-.4.0%, necrotic cells:
13.0.+-.2.1%, unclassified: 2.5.+-.1.3%, P<0.05; FIGS. 10A and
B). In contrast, in RIP-3-/- mice, Z-VAD treatment substantially
prevented photoreceptor death after retinal detachment without
inducing necrotic cell death (% apoptotic cells: 4.8.+-.1.3%,
necrotic cells: 6.4.+-.1.3%, unclassified: 0.9.+-.0.4%). FIG. 10A
shows TEM micrographs in the ONL on day three after retinal
detachment in WT and RIP-3-/- retina treated with vehicle, Nec-1 (a
Nec-1 of Formula 1-C), Z-VAD, or Nec-1+Z-VAD. In untreated attached
retina, the retinal morphology was similar in WT and RIP-3-/- mice
(A: apoptotic cell. N: Necrotic cell.) FIG. 10B shows the
quantification of apoptotic and necrotic photoreceptor death after
retinal detachment (n=4 each). RIP-3 deficiency inhibits the switch
to necrotic cell death by Z-VAD treatment and prevents
photoreceptor death after retinal detachment. Scale bar, 5 m.
[0412] Consistent with these results, the number of photoreceptors
with disrupted plasma membrane, as assessed by in vivo PI-labeling,
was not increased by Z-VAD treatment in RIP-3-/- retinas (FIGS. 8C
and 8D), confirming that RIP-3 plays an essential role to induce
programmed necrosis after retinal detachment, especially in the
presence of a caspase inhibitor. FIG. 8C-D show PI staining (FIG.
8C) and quantification of PI-positive photoreceptors (FIG. 8D) on
day 3 after retinal detachment in retina treated with vehicle,
Z-VAD or Nec-1 (a Nec-1 of Formula I-C) and Z-VAD in RIP-3-/-
retina (n=5-6; C and D). *, P<0.05; **, P<0.01. Scale bar,
100 .mu.m. In the WT mice, treatment with Z-VAD showed an increase
in the number of PI-positive photoreceptors compared to
vehicle-treated retina three days after retinal detachment
suggesting increased necrotic cell death in pan-caspase inhibited
photoreceptor cells. In comparison, treatment with Z-VAD in the
RIP-3-/- mice did not show an increase in the number of PI-positive
cells in the ONL indicating that there was no shift to necrotic
cell death in the RIP-3-/- cells following treatment with a
pan-caspase inhibitor after retinal detachment.
[0413] In addition, RIP-3-/- retinas showed less apoptotic cells
after retinal detachment (% apoptotic cells: 13.4.+-.1.1%, necrotic
cells: 8.1.+-.1.0%, unclassified: 1.2.+-.0.5%) compared to WT
retinas, suggesting that RIP-3 may be involved not only in
RIP-1-mediated programmed necrosis but also in other cell death
pathways. Alternatively, the kinetics of a single administration of
Nec-1 to inhibit RIP kinase is different than the chronic loss of
RIP kinase activity in RIP-3-/- animals.
[0414] These data indicate that RIP-3 mediates necrotic
photoreceptor death after retinal detachment. Suppression of RIP-3
gene expression significantly limits necrotic photoreceptor death
after retinal detachment via a similar pathway to Nec-1 treatment.
Treatment with Nec-1 does not provide additional protection in
RIP-3-/- mice. However, administration of Z-VAD in a RIP-3-/- mouse
significantly limits photoreceptor death after retinal detachment
suggesting that blocking both necrosis and apoptosis mediated cell
death may be used to prevent vision loss in various retinal
disorders associated with retinal detachment. Thus, RIP-3
deficiency provides neuroprotection after retinal detachment that
is augmented by Z-VAD. These results indicate that the suppression
of RIP-3 expression prevents a shift of retinal detachment-induced
photoreceptor death from apoptosis to programmed necrosis following
treatment with a pan-caspase inhibitor. These data further indicate
that RIP-3 mediates programmed necrosis and that blockade of the
RIP Kinase pathway is important and necessary in addition to
blockade of the caspase pathway for effective protection of
photoreceptor cells form cell death.
Example 5: RIP-1 Kinase Mediates Inflammatory Reactions after
Retinal Detachment
[0415] In this example, the role of RIP-1 kinase activity in
inflammatory reactions after retinal detachment was
investigated.
[0416] Immunohistochemistry (IHC) was performed as previously
reported (Murakami Y et al. (2008) AM J PATHOL 173:1326-1338). Rat
anti-CD11b antibody (1:50; BD Biosciences, San Diego, Calif.) was
used as the primary antibody. The specimens were imaged by confocal
microscopy using the Leica HCX APOL40X lens.
[0417] Immunofluorescence was performed as previously reported
(Hisatomi et al. (2008) J CLIN INVEST 118:2025-2038. The enucleated
eyes were frozen in OCT compound (Sakura Finetechnical Co., Tokyo,
Japan). Five .mu.m-thick sections were cut, air dried and fixed in
cold acetone for 10 minutes. Rat anti-CD11b (1:50; BD Biosciences,
San Diego, Calif.) was used as the primary antibody and incubated
at 4.degree. C. overnight. A nonimmune serum was used as a negative
control. Alexa Fluor 594-conjugated goat anti-rat IgG (Invitrogen,
Carlsbad, Calif.) was used as the secondary antibody and incubated
at room temperature for one hour. The specimens were imaged by
confocal microscopy using the Leica HCX APOL40X lens.
[0418] Total RNA extraction and reverse transcription were
performed as previously reported (Nakazawa T et al. (2006) MOL VIS
12:867-878; Hisatomi T et al. (2008). J CLIN INVEST 118:2025-2038).
A real-time PCR assay was performed with Prism 7700 Sequence
Detection System (Applied Biosystems, Foster City, Calif.). TaqMan
Gene Expression assays were also used to check the expression of
MCP-1 (Rn00580555_m1), TNF-.alpha. (Rn99999017_m1), and Fas-L
(Rn00563754_m1). For relative comparison of each gene, the Ct value
of real-time PCR data was analyzed with the AACt method and
normalized by an endogenous control (18S ribosomal RNA).
[0419] In addition, the MCP-1 protein contents in rat retina were
determined using ELISA Kit for rat MCP-1 (Thermo, Rockford,
Ill.).
[0420] Since the release of intracellular content in necrosis
results in secondary inflammation, changes in the inflammatory
response after retinal detachment were investigated by
immunohistochemistry and immunofluorescence with an antibody
against CD11b. FIG. 11A-B show IHC for CD11b (FIG. 11A) and
quantification of CD11b-positive cells in control retina without
retinal detachment and in retina treated with vehicle, Z-VAD or
Z-VAD+Nec-1 (n=6 each) (FIG. 11B) at three days after retinal
detachment. (*, P<0.05; **, P<0.01; NS, not significant;
Scale bar, 50 .mu.m). On day three after retinal detachment,
vehicle-treated detached retina showed infiltration of
CD11b-positive macrophage/microglia in the retina and subretinal
space (395.9.+-.83.8 cells/mm.sup.2; FIG. 11A-B). Eyes treated with
Z-VAD demonstrated even greater infiltration of CD11b-positive
cells in the retina (514.7.+-.54.5 cells/mm.sup.2) with clusters of
these cells present in the ONL (FIG. 11A-B), indicating that
necrotic cell death may promote inflammatory reaction. This
infiltration of CD11b-positive cells was significantly suppressed
with Z-VAD+Nec-1 treatment (262.9.+-.44.6 cells/mm.sup.2,
P<0.01; FIG. 11A-B). In comparison, significantly fewer CD11-b
cells infiltrated the retina and subretinal space of RIP-3-/- mice.
Further, RIP-3-/- eyes treated with Z-VAD demonstrated even less
infiltration of CD11-b positive cells in the retina and subretinal
space.
[0421] FIG. 11C-D show immunofluorescence for CD11b (FIG. 11C) and
quantification of CD11b-positive macrophage/microglia (FIG. 11D) in
WT and RIP-3-/- retina after retinal detachment. In WT mice, Z-VAD
treatment significantly increased infiltration of CD11b-positive
cells compared to vehicle treatment (P<0.05). This increase of
CD11b-positive cells was significantly suppressed with Nec-1+Z-VAD
treatment or RIP-3 deficiency (P<0.01; Scale bar, 50 .mu.m).
[0422] Although monocyte chemoattractant protein 1 (MCP-1) was
previously described as an essential mediator of early infiltration
of macrophage/microglia and the subsequent cell death after retinal
detachment (Nakazawa T et al. (2007) PROC NATL ACAD SCI USA
104:2425-2430), Z-VAD+Nec-1 combination treatment did not affect
MCP-1 expression (FIG. 12A and FIG. 13A). Further, FIG. 11E-F show
an ELISA for MCP-1 on day three after retinal detachment in either
WT or RIP-3-/- retinas (n=5 each; FIG. 11D) treated with vehicle
(n=5), Z-VAD (n=5) or Nec-1+Z-VAD (n=6; FIG. 11C). Retinas without
retinal detachment were used as controls. (*, P<0.05; **,
P<0.01; NS, not significant). RIP-3-/- retinas also showed
substantial upregulation of MCP-1 after retinal detachment,
although its expression level was slightly decreased than that in
WT retinas (FIG. 11F).
[0423] Cell death and inflammation communicate with each other
during neurodegeneration (Zitvogel et al. (2010) CELL 141:798-804).
Infiltrating inflammatory cells stimulate neuronal cell death
(Nakazawa et al. (2007) PROC NATL ACAD SCI USA 104:2425-2430), and
conversely dying cells, especially of the necrotic form, trigger
inflammation (Cavassani et al. (2008) J EXP MED 205:2609-2621). We
previously described that MCP-1 is a key mediator of early
infiltration of macrophage/microglia after retinal detachment
(Nakazawa et al. (2007) PROC NATL ACAD SCI USA 104:2425-2430).
However, MCP-1 upregulation after retinal detachment was not
substantially altered by Nec-1+Z-VAD combination treatment or by
RIP-3 deficiency, suggesting that RIP kinases are not involved in
the initiation of inflammation. The in situ hybridization data
described herein indicate that RIP-3 is detected in the ONL after
retinal detachment, suggesting that RIP kinase inhibition may
target photoreceptors and suppress inflammation subsequent to
photoreceptor death. However, since RIP-3 is known to be expressed
in several cell types including macrophages (Newton et al. (2004)
MOL CELL BIOL 24:1464-9), and given the limited resolution of our
in situ hybridization the possibility that RIP kinase inhibition
may affect macrophage/microglia function cannot be excluded;
defining the role of RIP kinases in cell death and inflammation
warrants further studies using tissue-specific RIP-3-/-
animals.
Example 6: RIP Kinases Mediate ROS Production and AIF Nuclear
Translocation after Retinal Detachment
[0424] The level of reactive oxygen species (ROS) production and
apoptosis-inducing factor (AIF) nuclear translocation were measured
after retinal detachment.
[0425] Immunohistochemistry (IHC) was performed as previously
reported (Murakami Y et al. (2008) AM J PATHOL 173:1326-1338).
Rabbit anti-AIF antibody (1:100; Cell Signaling Technology,
Danvers, Mass.) was used as the primary antibody. The specimens
were imaged by confocal microscopy using the Leica HCX APOL40X
lens.
[0426] Immunofluorescence was performed as previously reported
(Hisatomi et al. (2008) J CLIN INVEST 118:2025-2038. The enucleated
eyes were frozen in OCT compound (Sakura Finetechnical Co., Tokyo,
Japan). Five m-thick sections were cut, air dried and fixed in cold
acetone for 10 minutes. Rabbit anti-AIF antibody (1:100; Cell
Signaling Technology, Danvers, Mass.) was used as the primary
antibody and incubated at 4.degree. C. overnight. A non-immune
serum was used as a negative control. Alexa Fluor 488-conjugated
goat anti-rabbit IgG was used as the secondary antibody and
incubated at room temperature for one hour. The specimens were
imaged by confocal microscopy using the Leica HCX APOL40X lens.
[0427] Recent studies reported that RIP kinases regulate downstream
ROS production during programmed necrosis (Cho Y S et al. (2009)
CELL 137:1112-1123; Zhang D W et al. (2009) SCIENCE 325:332-336;
Kim et al. (2007) MOL CELL 26:675-687). ELISA for carbonyl adducts
of proteins was used to examine oxidative retinal damage following
retinal detachment. Specifically, the amounts of protein carbonyls
were determined by using OxiSelect.TM. Protein Carbonyl ELISA Kit
(Cell Biolabs, San Diego, Calif.). As shown in FIG. 14A, the
carbonyl contents in control retina without retinal detachment
(n=6-8) and in retina three days after retinal detachment with
treatment of vehicle, Z-VAD or Z-VAD+Nec-1 (the Nec-1 compound was
a compound of Formula I-C; n=7 each) were detected by an ELISA in
wild type (FIG. 14A) and in RIP-3-/- retina (n=10) (FIG. 14B). The
results indicate that on day three after retinal detachment, the
retina treated with vehicle or Z-VAD demonstrated significant
increase of carbonyl contents per mg protein compared to untreated
retina (P<0.05; FIG. 14A). In contrast, Z-VAD+Nec-1 treatment
almost completely suppressed the increase of carbonyl contents
after retinal detachment (P<0.05; FIG. 14A). RIP-3-/- retinas
also showed significantly less carbonyl contents after retinal
detachment compared to WT retinas (P<0.05; FIG. 14B).
[0428] Apoptosis-inducing factor (AIF) is a caspase-independent
inducer of cell death, which is released from the mitochondria and
translocates into the nuclei during cell death (Susin S A et al.
(1999) NATURE 397:441-446). The mitochondrial release of AIF is a
critical event for the photoreceptor death after retinal detachment
(Hisatomi T et al. (2001) AM. J. PATHOL. 158:1271-1278; Hisatomi T
et al. (2008) J. CLIN. INVEST. 118:2025-2038). Because ROS
overproduction is known to induce mitochondrial membrane
permeabilization and AIF release (Krantic S et al. (2007) PROG.
NEUROBIOL. 81:179-196), the cellular localization of AIF was
examined by IHC and immunofluorescence. The untreated retina showed
positive staining for AIF in the inner segment of photoreceptors,
which are a mitochondrion-rich region. FIG. 14C-F show double
staining for AIF and TUNEL (14C and E) and quantification of
AIF/TUNEL double positive photoreceptors (14D and F) in control
retina or in WT and RIP-3-/- retinas (n=5 each; FIGS. 14E and F)
treated with vehicle, Z-VAD or Nec-1+Z-VAD (the Nec-1 compound was
a compound of Formula I-C; n=6 each) three days after retinal
detachment (IS: inner segment. *, P<0.05; **, P<0.01; Scale
bar, 50 .mu.m). In vehicle- or Z-VAD-treated retina, AIF staining
was observed in TUNEL-positive photoreceptor nuclei
(AIF/TUNEL-double positive cells: 933.9.+-.210.6 cells/mm.sup.2 and
1021.9.+-.233.9 cells/mm.sup.2, respectively). In contrast,
Z-VAD+Nec-1 treatment significantly reduced AIF nuclear
translocation (281.3.+-.93.5 cells/mm.sup.2, P<0.01; FIG. 7B-C).
The data demonstrate that AIF translocation into TUNEL-positive
photoreceptor nuclei was increased after retinal detachment;
whereas Nec-1+Z-VAD combination treatment or RIP-3 deficiency
substantially reduced AIF nuclear translocation. These data also
demonstrate that ROS production and AIF nuclear translocation are
downstream events of RIP signaling.
[0429] In addition, overproduction of ROS has been implicated in
mitochondrial dysfunction and programmed necrosis (Vercammen D et
al. (1998) J. EXP. MED. 188:919-930; Lin Y et al. (2004) J. BIOL.
CHEM. 279:10822-10828). NADPH oxidase 1 has been shown to form a
complex with RIP-1 and generate superoxide during programmed
necrosis of L929 and MEF cells (Kim Y S et al. (2007) MOL. CELL
26:675-687). Alternatively, RIP-3 activates metabolic enzymes such
as glutamate dehydrogenase 1, and thereby increases mitochondrial
ROS production in NIH3T3 cells (Zhang et al. (2009) SCIENCE
325:332-336). Although there might be several molecular pathways
and cell sources for ROS production in vivo, the results disclosed
herein indicate that RIP-1 kinase mediates, at least in part, the
ROS generation after retinal detachment, and the mitochondrial
release of AIF, a key effector of caspase-independent apoptosis and
programmed necrosis (Susin S A et al. (2000) EXP. MED. 192:571-580;
Moubarak R S et al. (2007) MOL. CELL BIOL. 27:4844-4862). Further,
the results disclosed herein demonstrate that ROS production after
retinal detachment was suppressed by RIP kinase inhibition, i.e.,
inhibition of RIP-1 kinase by Nec-1 efficiently prevents ROS
production, AIF nuclear translocation, and necrotic photoreceptor
death after retinal detachments. In addition, RIP kinases act
upstream of AIF nuclear translocation. Recent studies also
demonstrate that AIF is an essential mediator of programmed
necrosis induced by alkylating DNA damage (Moubarak et al. (2007)
MOL CELL BIOL 24:4844-4862). These results suggest that RIP
kinases, AIF translocation, and programmed necrosis are linked.
[0430] The data suggests that RIP-1 is an adaptor kinase that acts
downstream of death domain receptors and is essential for both cell
survival and death (Vandenabeele et al. (2010) SCI SIGNAL 3: re4).
The intermediate domain of RIP-1 mediates activation of the
prosurvival transcriptional factor NF-.kappa.B through
polyubiquitination of RIP-1 (Li H et al. (2006) J. BIOL. CHEM.
281:13636-13643), and RIP-1 knockout mice die soon after birth with
reduced NF-icB activation and massive necrosis of lymphoid and
adipose tissue (Kelliher M A et al. (1998) IMMUNITY 8:297-303). On
the other hand, the N-terminal kinase functions of RIP-1 is crucial
for programmed necrosis but dispensable for prosurvival of
NF-.kappa.B activation (Holler N et al. (2000) NAT. IMMUNOL.
1:489-495; Degterev A et al. (2008) NAT. CHEM. BIOL. 4:313-321; Lee
T H et al. (2004) J. BIOL. CHEM. 279:33185-33191). Consistent with
these results, Nec-1 showed no effect on NF-.kappa.B p65
phosphorylation after retinal detachment (FIG. 16). RIP-1 switches
function to a regulator of cell death when it is unubiquitinated
and forms a death-signaling complex (Mahoney et al. 2008 PROC.
NATL. ACAD. SCI. USA 105:11778-11783; Wang et al. (2008) CELL
133:693-703). In addition, recent studies revealed that formation
of RIP-1-RIP-3 complex is a critical step in the RIP-1 kinase
activation and induction of programmed necrosis (He et al. (2009)
CELL 137:1100-1111; Cho et al. (2009) CELL 137:1112-1123; Zhang et
al. (2009) SCIENCE 325:332-336). Further, He et al. showed that
RIP-1 was expressed at similar levels in fourteen cell lines
undergoing cell death, while RIP-3 expression levels correlated
with the cellular necrotic response (He et al. (2009) CELL
137:1100-1111). After retinal detachment, RIP-3 expression
increased over 10-fold and RIP-3 deficiency prevented the shift to
necrotic photoreceptor death by Z-VAD suggesting that RIP-3 is a
key regulator of programmed necrosis after retinal detachment.
Thus, the greater fold increase of RIP-3 compared to RIP-1 that was
observed after retinal detachment may be a crucial step that
directs the photoreceptors to programmed necrosis.
Example 7: Suppression of Macroautophagy Induction in the Retina
after Retinal Detachment
[0431] The level of macroautophagy induction in the retina was
measured following retinal detachment. Cell death with autophagy is
another form of cell death that is morphologically defined by the
lack of chromatin condensation and accumulation of autophagic
vacuoles (Kroemer G et al. (2009) CELL DEATH DIFFER. 16:3-11), and
these morphological signs have been described in several
neurodegenerative diseases (Kourtis N & Tavernarakis N (2009)
CELL DEATH DIFFER. 16:21-30). Degterev et al. also reported that
macroautophagy was activated during programmed necrosis (2005 NAT.
CHEM. BIOL. 1:112-119).
[0432] The morphological change of photoreceptors after retinal
detachment was analyzed by TEM, but photoreceptors associated with
autophagic vacuoles were not apparent in each group.
[0433] For Western Blot analysis, the vitreous and neural retina
combined, were collected at three days after retinal detachment.
Samples were run on 4-12% SDS-polyacrylamide gel electrophoresis
and transferred onto PVDF membrane. After blocking with 3% nonfat
dried milk, the membrane was reacted with anti-light chain 3
antibody (1:1000; Cell signaling), anti-phosphorylated ribosomal
protein S6 antibody (1:1000; Cell Signaling) or anti-phosphorylated
NF-.kappa.B p65 antibody (1:1000; Cell Signaling). Immunoblots were
then developed with enhanced chemiluminescence. Lane-loading
differences were normalized by .beta.-tubulin (1:1000; Cell
Signaling).
[0434] Western blot analysis of the lipidated form of light chain
3, a well-known marker of macroautophagy, demonstrated no
significant difference in its expression after retinal detachment
(FIG. 17A). Phosphorylated ribosomal protein S6, reflecting
activity of ribosomal S6 kinase 1, an inhibitor of macroautophagy,
was substantially upregulated in the retina after retinal
detachment (FIG. 17B). These data indicate that macroautophagy
induction may be suppressed in the retina after retinal
detachment.
[0435] In addition, chaperone-mediated autophagy (CMA), another
lysosomal degradation pathway, is known to be upregulated during
starvation and oxidative stress (Dice J F (2007) AUTOPHAGY
3:295-299). Lysosomal-associated membrane protein type 2A
(LAMP-2A), a lysosomal receptor involved in CMA, is a marker of
CMA. Starvation increases LAMP-2A by preventing its degradation
while oxidative stress results in transcriptional upregulation of
LAMP-2A. However, LAMP-2A expression levels were unchanged in the
retina after retinal detachment, although its localization in
lysosomal membrane that directly correlates with CMA activity
remains to be addressed in further studies (Cuervo A M & Dice J
F (1996) SCIENCE 273:501-503).
Example 8: Z-VAD+Nec-1 Treatment was More Effective than
TNF-.alpha. Blockage
[0436] TNF-.alpha. and Fas-L are potent inducer of programmed
necrosis as well as apoptosis (Degterev A (2005) NAT CHEM BIOL
1:112-119). The effectiveness of a TNF-.alpha. blockade after
retinal detachment was measured. In particular, the role of
TNF-.alpha., one of the main stimuli responsible for the initiation
of programmed necrosis of photoreceptors after retinal detachment,
was investigated in part because TNF-.alpha. was increased in
detached retina (FIGS. 18A and 18B) and treatment with a
neutralizing anti-TNF-.alpha. antibody suppressed photoreceptor
loss (FIG. 18C-D). However, other factors may also contribute to
photoreceptor death, as the effect of TNF-.alpha. blockade was not
as effective as that of Nec-1+Z-VAD treatment. Further, Fas ligand
expression increased in Z-VAD-treated retina after retinal
detachment compared to vehicle treatment (FIG. 12C), and may play a
role in promoting necrotic photoreceptor death in Z-VAD-treated
eyes.
[0437] TNF-.alpha. protein contents in rat retina were determined
using ELISA Kit for rat TNF-.alpha. (Invitrogen, Carlsbad, Calif.).
Goat anti-mouse/rat TNF-.alpha. blocking antibody was purchased
from R&D Systems (Minneapolis, Minn.). The rat eyes were
subretinally injected with 1% of sodium hyaluronate containing 0.1
mg/ml of anti-TNF-.alpha. antibody or control antibody.
[0438] The results indicate that TNF-blockade was not as effective
as treatment with Z-VAD in combination with Nec-1. After retinal
detachment, TNF-.alpha. was increased in detached retina (FIGS. 18A
and 18B) and treatment with a neutralizing anti-TNF-.alpha.
antibody suppressed photoreceptor loss (FIG. 18C), suggesting that
TNF-.alpha. may contribute to the induction of apoptosis and
programmed necrosis. In addition to TNF-.alpha., Fas-L/Fas pathway
is known to be activated and mediate photoreceptor death after
retinal detachment (Zacks et al. (2004) INVEST OPHTHALMOL VIS SCI
45:4563-4569; Zacks et al. (2007) ARCH OPTHALMOL 125:1389-1395),
and may cooperate with TNF-.alpha. to activate RIP kinase and
promote programmed necrosis in addition to apoptosis. (FIG. 19). As
shown in FIG. 19, RIP-1 mediates prosurvival NF-.kappa.B activation
through polyubiquitination of RIP-1 in response to TNF-.alpha..
When RIP-1 is unubiquitinated, RIP-1 switches function to a
regulator of cell death. RIP-1 forms a death signaling complex with
Fas-associated death domain (FADD) and caspase-8 after stimulation
of death domain receptors, and induces caspase-dependent apoptosis.
In conditions where caspase pathway is blocked, RIP-1 kinase is
activated in RIP-1-RIP-3 complex and promotes programmed necrosis.
TRAF2: tumor necrosis factor-receptor associated signaling adaptor.
TRADD: TNF receptor-associated death domain. cIAP: cellular
inhibitor of apoptosis. CYLD: cylindromatosis. Thus, RIP Kinases
act as common intermediaries for various upstream death signals and
their blockade in addition to caspases is likely necessary for
effective neuroprotection.
Example 9: Efficacy of a Necrosis Inhibitor and a Pan-Caspase
Inhibitor in the Treatment of AMD
[0439] A. Increased Expression of RIP-3 and RIP-1 in a Poly I:C
Induced AMD Model
[0440] In this example, the mechanism of photoreceptor cell death
in dry AMD was investigated using a polyinosinic:polycytidylic acid
(Poly I:C)-induced dry AMD model (Yang et al. (2009) NEJM
359(14):1456-63). Poly I:C is a synthetic double-stranded RNA
(dsRNA) analog that activates toll-like receptor 3 (TLR3) signaling
(FIG. 20A). Furthermore, dsRNA are known to exist in the drusen of
human AMD patients. As depicted in FIG. 20A, activation of the TLR3
signaling results in activation of RIP-1 kinase pathway. FIG. 20B
also shows that intraocular injection of poly I:C in mice caused
significant geographic loss of photoreceptors and RPE thus
mimicking human dry AMD (compare to PBS treated mice). In the
experiments described herein, 2-3 .mu.l of Poly I:C (1 mg/ml) was
injected into the subretinal space of the eye (e.g., approximately
2-3 .mu.g of Poly I:C was administered to the eye) to induce
AMD.
[0441] Pathways leading to photoreceptor death in AMD were
investigated. In particular, the expression levels of RIP-1 and
RIP-3 proteins were measured in the Poly I:C-induced dry AMD model.
RIP-3 is a key regulator of RIP-1 kinase activation (Galluzzi L et
al. (2009) J. MOL. CELL BIOL.) and its expression level has been
shown to correlate with responsiveness to programmed necrosis (He
et al. (2009) CELL 137:1100-1111). As described below, it was
demonstrated that necrotic photoreceptor death occurs in dry
AMD.
[0442] Total RNA extraction and reverse transcription were
performed as previously reported (Nakazawa T et al. (2006) MOL.
VIS. 12:867-878) (Hisatomi T et al. (2008). J. CLIN. INVEST.
118:2025-2038). A real-time PCR assay was performed with Prism 7700
Sequence Detection System (Applied Biosystems, Foster City,
Calif.). TaqMan Gene Expression assays were used to check the
expression of RIP-1 (Rn01757378_m1) and RIP-3 (Rn00595154_m1). For
relative comparison of each gene, the Ct value of real-time PCR
data was analyzed with the AACt method and normalized by an
endogenous control (18S ribosomal RNA).
[0443] The expression levels of RIP-3 and RIP-1 mRNA were assessed
in the retina by quantitative real-time PCR. FIG. 21A-B provide
graphs showing the quantitative real-time PCR analysis for RIP-1
(FIG. 21A) and RIP-3 (FIG. 21B) in control retina (untreated) and
in the retina of mice treated with poly I:C. RIP-1 expression
increased approximately two-fold in the retina of mice treated with
poly I:C compared to that in untreated retina (FIG. 21A). RIP-3
expression increased approximately 6-fold in poly I:C treated mice
compared to control (FIG. 21B). The expression levels of RIP-3 in
RPE cells and macrophages were also examined. As depicted in FIG.
21C, RIP-3 expression increased approximately three-fold in the RPE
cells of mice treated with poly I:C compared to that in untreated
RPE cells. In comparison, no significant difference in RIP-3
expression was observed in macrophage cells in mice treated with
poly I:C compared to controls (FIG. 21D).
[0444] These data suggest that RIP-mediated programmed necrosis is
involved in photoreceptor loss in dry AMD.
[0445] B. RIP-3 Deficiency Prevents Poly I:C-Induced Retinal
Degeneration
[0446] The role of RIP-3 in RIP-mediated programmed necrosis in
photoreceptor loss in AMD was investigated using RIP-3-/- mice.
TUNEL assays were used to measure the amount of apoptotic and
necrotic cell death (Grasl-Kraupp et al., HEPTALOGY (1995)
21(5):1465-8). TUNEL and quantification of TUNEL positive cells in
the ONL were performed as previously described (See Example 2). The
thickness of the ONL was determined by the National Institutes of
Health ImageJ software. FIG. 22A shows TUNEL (green) and DAPI
(blue) staining in retina from wild type or RIP-3-/- mice treated
with poly I:C. Two days after poly I:C treatment, RIP-3-/- mice
showed significantly less TUNEL-positive cells than WT mice.
Specifically, the number of TUNEL-positive cells in the RIP-3-/-
mice treated with poly I:C was at least three fold less than
control mice consistent with RIP-3 playing a role in mediating
programmed necrosis in AMD (FIG. 22B).
[0447] The thickness of the outer nuclear layer (ONL) was also
determined in WT or RIP-3-/- mice at 14 days following treatment
with poly I:C. Sections were randomly selected from either WT or
RIP-3-/- mice following poly I:C treatment and the retina was
photographed. The ONL thickness was measured in each section by
masked observers. FIG. 22C-D show that ONL thickness was
significantly reduced in WT retina treated with poly I:C. In
comparison, ONL thickness was not affected in RIP-3-/- retina
treated with poly I:C indicating that RIP-3 deficiency had a
protective effect in photoreceptor loss in AMD.
[0448] Photoreceptors in WT or RIP-3-/- mice following poly I:C
treatment were also examined by PI staining. In vivo PI staining
was performed by injecting 5 .mu.l of PI (50 .mu.g/ml) into the
subretinal space at two days after poly I:C treatment. After two
hours, the eyes were enucleated and 10 m thick cryosections were
cut, air-dried, and fixed in 100% ethanol. DAPI was used to
counterstain the nuclei. The center of the retina was photographed
by fluorescence microscope, and the number of PI-positive cells in
ONL was analyzed by ImageJ software.
[0449] FIG. 23A-B show PI staining (FIG. 23A) and quantification of
PI-positive photoreceptors (FIG. 23B) on day two after treatment
with vehicle or poly I:C in wildtype or RIP-3-/- mice. Subretinal
injection of PI prior to enucleation, a technique to demonstrate
cells with disrupted cell membrane, showed decreased number of
PI-positive photoreceptors in RIP-3-/- mice compared to wildtype
mice. These findings are consistent with the TUNEL and ONL
thickness studies, and indicate a involvement of RIP-mediated
programmed necrosis in dry AMD.
[0450] C. Necrosis Inhibitor Prevents Poly I:C-Induced Retinal
Degeneration in Combination with a Caspase Inhibitor
[0451] Necrostatin-1 is a potent and selective inhibitor of
programmed necrosis, which targets RIP-1 kinase activity (Degterev
A et al. (2008) NAT. CHEM. BIOL. 4:313-321). The effectiveness of a
RIP-1 kinase inhibitor in combination with a caspase inhibitor in
the treatment of dry AMD was investigated.
[0452] To determine the effect of Nec-1 and a pan-caspase inhibitor
in the treatment of AMD, TUNEL assay was performed to assess
photoreceptor cell death in the poly I:C induced AMD model. FIG.
24A shows that treatment with Z-VAD or Nec-1 alone had a slight
effect on the number of TUNEL-positive cells in ONL as compared
with vehicle treatment. In contrast, combined treatment with
Nec-1+Z-VAD significantly reduced the number of TUNEL-positive
cells in ONL. The thickness of ONL was also measured. As shown in
FIG. 24B, the combination of Nec-1+Z-VAD significantly prevented
the reduction in ONL thickness, whereas either Z-VAD or Nec-1 alone
had minimal effect in preserving retinal thickness.
[0453] Collectively, these results indicate that programmed
necrosis as well as apoptosis is involved in photoreceptor death in
AMD, and that RIP-mediated programmed necrosis becomes a
predominant form of photoreceptor death when caspase-dependent
apoptotic pathway is inhibited.
[0454] D. Increased RIP-1 and RIP-3 Expression in the
CX3CR1-/-CCL2-/- Mouse Model of AMD
[0455] CX3CR1-/-CCL2-/- double knockout mice is a well-established
model for dry and wet AMD (Tuo et al. IOVS (2007) 48(8):
3827-3836). At 9-12 weeks of age, these mice spontaneously develop
drusen-like lesions characterized by heterogeneous, round or
domed-shaped, soft-bordered, yellowish deposits within the
subretina (FIG. 25A), which are associated with dry AMD. By 6
months of age, choroidal neovascularization, a hallmark of wet AMD,
was observed in the eye of the CX3CR1-/-CCL2-/- double knockout
mice (FIG. 25A).
[0456] The expression levels of RIP-1 and RIP-3 mRNA were assessed
in the retina of CX3CR1-/-CCL2-/- double knockout mice at 5 weeks
(5 w) and at 5 months (5M) of age. As shown in FIG. 25B, RIP-1
expression was elevated in the CX3CR1-/-CCL2-/- double knockout
mice compared to WT mice. Further, RIP-1 expression progressively
increased with age. Similarly, RIP-3 expression was also elevated
in CX3CR1-/-CCL2-/- double knockout mice compared to WT mice. In
addition, RIP-3 expression also increased with age (FIG. 25C).
These results are consistent with results from the poly I:C-induced
AMD model indicating that RIP-mediated programmed necrosis is
involved photoreceptor death in AMD.
[0457] Together, the data from both the poly I:C induced and
CX3CR1-/-CCL2-/- double knockout AMD models indicate that RIP-3
mediates necrotic photoreceptor death in AMD. To further confirm
the role of RIP-3 mediated necrosis in AMD, CX3CR1/CCL2/RIP-3
triple knockout mice were generated. As described previously,
CX3CR1-/-CCL2-/- double knockout mice spontaneously develop
drusen-like lesions within the subretina by 9-12 weeks of age. In
comparison, CX3CR1-/-CCL2-/-RIP-3-/- mice exhibited a significant
reduction in the number of drusen-like lesions within the subretina
(FIG. 26).
[0458] Collectively, these data indicate that suppression of RIP-3
gene expression significantly reduces the clinical features of AMD.
These data further indicate that blockade of the RIP kinase pathway
is important and necessary in addition to blockade of the caspase
pathway for effective protection of photoreceptor cells in AMD.
[0459] E. RIP-3 Deficiency Reduces Inflammatory Cytokine Expression
in the CX3CR1-/-CCL2-/- Mouse Model of AMD
[0460] The expression levels of various inflammatory cytokines were
assessed in the retina of CX3CR1-/-CCL2-/- double knockout mice at
2 months (2M) of age. As shown in FIG. 27, the expression of
certain inflammatory cytokines such as TNF-.alpha., CCL3
(MIP1-alpha), CCL5 (RANTES), and IL-6 were increased in the
CX3CR1-/-CCL2-/- double knockout mice compared to WT mice. In
comparison, RIP-3 deficiency in the context of the
CX3CR1-/-CCL2-/-RIP-3-/- mice reduced the expression of these
inflammatory cytokines. This data indicates that RIP-3 is involved
in the inflammatory response associated with AMD. This data further
suggests that inhibition of RIP-3 may provide effective protection
against secondary inflammation associated with AMD.
Example 10: Efficacy of a Necrosis Inhibitor and a Pan-Caspase
Inhibitor in the Treatment of Retinitis Pigmentosa
[0461] Retinal degeneration 10 (rd10) mice are a model of autosomal
recessive retinitis pigmentosa (RP), identified by Chang et al. in
2002 (Vision Res. 42:517-525). These mice carry a spontaneous
mutation of the rod-phosphodiesterase (PDE) gene, leading to a rod
degeneration that starts around post-natal day 18 (P18) and
photoreceptor death which peaks at P25 (FIG. 28).
[0462] A. Increased Expression of RIP-3 and RIP-1 in Rd10 Mice
[0463] To investigate the role of RIP-mediated programmed necrosis
in RP, the expression levels of RIP-1 and RIP-3 mRNA were assessed
in rd10 mice. As shown in FIG. 29A, RIP-1 expression was
significantly elevated in rd10 mice compared to wild type mice and
peaked at P28. The slight drop in RIP-1 expression by P35 was most
likely due to significant death of photoreceptor cells by that
time. RIP-3 expression was also elevated in rd10 mice when compared
to control and its expression also peaked at P28 (FIG. 29B). These
data suggest that RIP-mediated programmed necrosis is involved in
photoreceptor loss in RP.
[0464] B. Simultaneous Inhibition of RIP Kinases and Caspases
Prevented Photoreceptor Cell Death in rd10 Mice
[0465] To determine the effect of Nec-1 and a pan-caspase inhibitor
in the treatment of RP, ONL thickness was measured to assess
photoreceptor cell death in rd10 mice. Mice were injected with
Nec-1 (50 .mu.g/g/day) and Z-VAD (10 .mu.g/g/day). At postnatal day
25 (P25) through P28, mice received daily intraperiotoneal
injections. As shown in FIG. 30A-B, the combination of Nec-1+Z-VAD
significantly prevented the reduction in ONL thickness in rd10 mice
compared to vehicle treated mice, indicating that RIP-mediated
necrosis as well as apoptosis are involved in photoreceptor cell
death in RP.
[0466] C. RIP-3 Deficiency Inhibits Induction of Programmed
Necrosis and Prevents Photoreceptor Death in RP.
[0467] The role of RIP-3 in RIP-mediated programmed necrosis in
photoreceptor death in RP was investigated in RIP-3 knockout mice.
ONL thickness was measured at postnatal day 28 (P28) to assess
photoreceptor cell death in rd10 RIP-3 double mutant mice. As shown
in FIG. 31A-B, RIP-3 deficiency significantly prevented the
reduction in ONL thickness in rd10 mice consistent with RIP-3
playing a role in mediating programmed necrosis in RP.
[0468] Collectively, these data indicate that programmed necrosis
and apoptosis are involved in photoreceptor loss in RP, and that
RIP-3 mediates necrotic photoreceptor death in this retinal
disorder. Suppression of RIP-3 gene expression significantly limits
necrotic photoreceptor death in RP via a similar pathway to Nec-1
treatment. These data further indicate that blockade of the RIP
kinase pathway is important and necessary in addition to blockade
of the caspase pathway for effective protection of photoreceptor
cells in RP.
Example 11: Increased Expression of RIP-1 and RIP-3 in RPE Cells
Following Poly I:C Treatment
[0469] As described previously in Example 9, poly I:C is a dsRNA
analog that activates TLR3 signaling and the RIP-1 kinase pathway.
Further, intraocular injection of poly I:C in mice caused
significant geographic loss of photoreceptors and RPE cells thus
mimicking human dry AMD (FIG. 20B). In this Example, pathways
leading to RPE cell death following poly I:C treatment were
investigated.
[0470] Primary RPE cells were extracted from the retina of mice and
cultured in the presence of 5 .mu.g/ml of poly I:C for 48 hours or
with PBS. Total RNA extraction and reverse transcription were
performed as previously reported (Nakazawa T et al. (2006) MOL.
VIS. 12:867-878) (Hisatomi T et al. (2008). J. CLIN. INVEST.
118:2025-2038). A real-time PCR assay was performed with Prism 7700
Sequence Detection System (Applied Biosystems, Foster City,
Calif.). TaqMan Gene Expression assays were used to check the
expression of RIP-1 (Rn01757378_m1) and RIP-3 (Rn00595154_m1). For
relative comparison of each gene, the Ct value of real-time PCR
data was analyzed with the AACt method and normalized by an
endogenous control (18S ribosomal RNA).
[0471] The expression levels of RIP-1 and RIP-3 mRNA were assessed
in primary RPE cells treated with poly I:C. FIG. 32A-B provide
graphs showing the quantitative real-time PCR analysis for RIP-1
(FIG. 32A) and RIP-3 (FIG. 32B) in control RPE cells treated with
PBS and in RPE cells after treatment with poly I:C. RIP-1
expression increased approximately two-fold in RPE cells treated
with poly I:C compared to untreated RPE cells. RIP-3 expression in
RPE cells also increased approximately three-fold after poly I:C
treatment.
[0472] These data indicate that RIP-mediated programmed necrosis is
involved in RPE cell loss following poly I:C treatment.
Example 12: Necrosis Inhibitor or RIP-3 Deficiency Prevents Poly
I:C Induced RPE Cell Death
[0473] In this Example, the effectiveness of a necrosis inhibitor
(i.e., necrostatin-1) to prevent poly I:C induced RPE cell death
was investigated.
[0474] As described in Example 11, primary RPE cells were extracted
from the retina of mice and cultured in the presence of 5 .mu.g/ml
of poly I:C for 48 hours or with PBS. Cell viability was measured
using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan).
This assay is based on the cleavage of
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-te-
trazolium, monosodium salt (WST-8) by the mitochondrial
dehydrogenase enzyme to produce a formazan dye. After incubation
with WST-8 for two hours at 37.degree. C., absorbance was measured
at 450 nm to determine cell viability.
[0475] FIG. 33A shows the viability of RPE cells treated with poly
I:C in combination with Z-VAD or Nec-1+Z-VAD. Z-VAD or Nec-1
treatment alone did not protect RPE cells from poly I:C induced
cell death. In comparison, Nec-1 combined with Z-VAD substantially
restored cell viability and prevented poly I:C induced cell death.
This data indicates that programmed necrosis as well as apoptosis
are involved in poly I:C induced RPE cell death, and that the RIP-1
and RIP-3 mediated programmed necrosis becomes a predominant form
of RPE cell death when the caspase-dependent pathway is
inhibited.
[0476] The role of RIP-3 in RIP-mediated programmed necrosis in RPE
cells was further investigated using poly I:C treated RIP-3-/-
cells. FIG. 33B shows the viability of WT or RIP-3-/- RPE cells
treated with poly I:C in combination with Z-VAD. Without poly I:C
treatment, the viability of RPE cells was similar in WT and
RIP-3-/- cells. Z-VAD treatment did not protect WT RPE cells from
poly I:C induced cell death. In contrast, RIP-3-/- cells treated
with both poly I:C and Z-VAD were protected from cell death.
[0477] Further, as described in Example 9, CX3CR1-/-CCL2-/-RIP-3-/-
mice exhibited a significant reduction in the number of drusen-like
lesions within the subretina when compared to CX3CR1-/-CCL2-/- mice
(FIG. 26). Drusen is usually located between the RPE cells and
their vascular supply, the choriocapillaris. The presence of drusen
may therefore interfere with RPE function by depriving the RPE
cells of oxygen and nutrients. Additionally, drusen formation is
thought to signify RPE dysfunction (i.e., result as a function of
diseased RPE). Our data that RIP-3 deficiency reduces drusen-like
lesions in the context of CX3CR1-/-CCL2-/-RIP-3-/- mice provides
further evidence that blockade of the RIP-3 kinase pathway may have
a protective effect on RPE function.
[0478] Collectively, these results indicate that programmed
necrosis as well as apoptosis are involved in poly I:C induced RPE
cell death. Without wishing to be bound by theory, it is
contemplated that blockage of the RIP kinase pathway in addition to
blockage of the caspase pathway, is necessary for effective
protection of RPE cell death.
Example 13: Efficacy of a Necrosis Inhibitor and/or an Apoptosis
Inhibitor in the Treatment of Retinal Detachment in Human
Subjects
[0479] The efficacy of a necrosis inhibitor (e.g., necrostain-1 or
necrostatin-4) and/or an apoptosis inhibitor (e.g., a pan-caspase
inhibitor, e.g., IDN-6556) in the treatment of retinal detachment
will be tested in human subjects.
[0480] Specifically, a necrostatin-1 or necrostatin-4 either alone
or in combination with IDN-6556 will be administered during one of
three standard surgical procedures for treating patients over the
age of 18 with a macula-off retinal detachment. There are
approximately 25,000 new patients diagnosed with macula-off retinal
detachment each year in the United States, with the average age of
these patients being in the 50s. The onset of the disease is fairly
rapid, and patients usually present within a few days to the eye
doctor with photopsias (light sensation), floaters and rapid vision
loss. The only treatment option for macula-off retinal detachment
is surgery, and currently there is no pharmacotherapy to prevent
photoreceptor cell loss in these patients. The three types of
surgical procedures used to treat macula-off retinal detachment are
pneumatic retinopexy, scleral buckle and vitrectomy.
[0481] For those patients treated with pneumatic retinopexy,
intravitral injections of the necrosis inhibitor (e.g.,
necrostatin-1 or necrostatin-4) either alone or in combination with
the apoptosis inhibitor (e.g., IDN-6556) will be administered
before the surgery (e.g., at the time of diagnosis or at least one
day before the surgery), at one week post-surgery, and possibly at
two weeks post-surgery. In some cases, the necrosis inhibitor
either alone or in combination with an apoptosis inhibitor will
also be administered at the time of surgery. It is contemplated
that 15 mM of IDN-6556 and/or 20 mM of necrostain-1 or
necrostatin-4 (in a total volume of 100 .mu.L) can be administered
to the patient to achieve a final concentration of 30 .mu.M and 400
.mu.M, respectively, in the eye (assuming that the average volume
of an eye is approximately 5 mL).
[0482] For patients treated with scleral buckle, intravitral
injections of the necrosis inhibitor (e.g., necrostatin-1 or
necrostatin-4) either alone or in combination with the apoptosis
inhibitor (e.g., IDN-6556) will be administered before the surgery
(e.g., at the time of diagnosis or at least one day before the
surgery), at the time of the surgery, at one week post-surgery, and
possibly at two weeks post-surgery.
[0483] For patients treated with vitrectomy, intravitral injections
of the necrosis inhibitor (e.g., necrostatin-1 or necrostatin-4)
either alone or in combination with the apoptosis inhibitor (e.g.,
IDN-6556) will be administered before the surgery (e.g., at the
time of diagnosis or at least one day before the surgery), at the
time of the surgery, at one week post-surgery, and possibly at
two-weeks post-surgery.
[0484] Patients who undergo surgical intervention only will serve
as controls. The study endpoint measures the proportion of patients
achieving 20/40 vision or better at three months after treatment.
It is contemplated that a higher proportion of patients treated
with a combination of a necrosis inhibitor (e.g., necrostatin-1 or
necrostatin-4) and an apoptosis inhibitor (e.g., IDN-6556) in
addition to surgery will achieve a 20/40 reading vision or better
and/or will show an improvement in levels of visual acuity gain
when compared to controls.
Example 14: Efficacy of a Necrosis Inhibitor and/or an Apoptosis
Inhibitor in the Treatment of Neovascular AMD in Human Subjects
[0485] The efficacy of a necrosis inhibitor (e.g., a necrostain-1
or necrostatin-4) and/or an apoptosis inhibitor (e.g., a
pan-caspase inhibitor, e.g., Z-VAD or IDN-6556) in the treatment of
AMD will be tested in human subjects.
[0486] Specifically, a necrostatin-1 or necrostatin-4 either alone
or in combination with IDN-6556 will be administered in combination
with anti-VEGF therapy for the treatment of patients over the age
of 18 who are affected with wet AMD. It is contemplated that 15 mM
of IDN-6556 and/or 20 mM of necrostain-1 or necrostatin-4 (in a
total volume of 100 .mu.L) can be administered to the patient to
achieve a final concentration of 30 .mu.M and 400 .mu.M,
respectively, in the eye (assuming that the average volume of an
eye is approximately 5 mL). Alternatively, a slow release
formulation of the necrosis inhibitor either alone or in
combination with an apoptosis inhibitor may be administered.
[0487] For those patients treated anti-VEGF therapy, intravitral
injections of the necrosis inhibitor (e.g., necrostatin-1 or
necrostatin-4) either alone or in combination with the apoptosis
inhibitor (e.g., IDN-6556) will be administered at the time of
diagnosis, at one-month, three-months, six-months, and possibly
twelve-months after the initial injection. Patients who receive
anti-VEGF injections alone will serve as controls.
[0488] The study endpoint measures the proportion of patients
achieving a 20/40 vision or better at six months and at twelve
months after the first injection of necrostatin-1 or necrostatin-4
alone or in combination with IDN-6556. It is contemplated that a
higher proportion of patients treated with a combination of a
necrostatin-1 and IDN-6556 or a combination of necrosatin-4 and
IDN-6556 in addition to anti-VEGF therapy will achieve a 20/40
reading vision or better and/or will show an improvement in levels
of visual acuity gain when compared to controls.
Example 15: Efficacy of a Necrosis Inhibitor and/or an Apoptosis
Inhibitor in the Treatment of Ocular Disorders in Human
Subjects
[0489] The efficacy of a necrosis inhibitor (e.g., a necrostain-1
or necrostatin-4) and/or an apoptosis inhibitor (e.g., a
pan-caspase inhibitor, e.g., IDN-6556) as an adjunct to the
treatment of the underlying ocular disorder will be tested in human
subjects. The effectiveness of combination therapy can be measured
in patients with an ocular disorder selected from retinitis
pigmentosa, Stargardt's disease, retinopathies (e.g., inflammatory
retinopathy, infectious retinopathy, and infectious retinopathy),
dry AMD, myopic degeneration, diabetic retinopathy, macular edema,
and central serous retinopathy. Specifically, a necrostatin-1 or
necrostatin-4 either alone or in combination with Z-VAD or IDN-6556
will be administered for the treatment of patients affected with
these ocular disorders.
[0490] For patients affected with retinitis pigmentosa, efficacy of
treatment may be measured by determining whether there is an
increase or preservation in the thickness of the macula as measured
by optical coherence tomography (OCT); a preservation or an
improvement of visual field (e.g., by at least 10% in the mean
standard deviation on the Humphrey Visual Field Test; and/or an
improvement or preservation of an electroretinograph (ERG), a
measurement of the electrical response of the retina to light
stimulation, (e.g., to increase ERG amplitude by at least 15%).
[0491] For patients affected with Stargardt's disease, efficacy of
treatment may be measured by determining whether there is a
preservation or an increase in the thickness of the macula as
measured by OCT; and/or an improvement or preservation of a
multifocal ERG.
[0492] For patients affected with Cone-Rod Dystrophy, efficacy of
treatment may be measured by determining whether there is an
increase in the thickness of the macula as measured by OCT; an
improvement of an ERG, and/or preservation of a multifocal ERG.
[0493] For patients affected with dry AMD with geographic atrophy,
efficacy of treatment may be evaluated by microperimetry and by
measurement of fundus autofluorescence. Alternatively, visual
acuity of these patients may be assessed.
INCORPORATION BY REFERENCE
[0494] The entire disclosure of each of the patent documents and
scientific articles cited herein are incorporated by reference in
their entirety for all purposes.
EQUIVALENTS
[0495] The invention can be embodied in other specific forms with
departing from the essential characteristics thereof. The foregoing
embodiments therefore are to be considered illustrative rather than
limiting on the invention described herein. The scope of the
invention is indicated by the appended claims rather than by the
foregoing description, and all changes that come within the meaning
and range of equivalency of the claims are intended to be embraced
therein.
Sequence CWU 1
1
57119PRTArtificial Sequencesource/note="Description of Artificial
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peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(19)..(19)Asp-aldehyde 1Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Pro Tyr1 5 10 15Val
Ala Asp220PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(20)..(20)Asp-aldehyde 2Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Asp
Glu Val Asp 20320PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(20)..(20)Pro-aldehyde 3Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Leu
Glu Val Pro 20420PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(20)..(20)Asp-aldehyde 4Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Val
Glu Ile Asp 20520PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(20)..(20)Asp-aldehyde 5Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Ile
Glu Thr Asp 20620PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AlaMOD_RES(20)..(20)Asp-aldehyde 6Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro1 5 10 15Leu
Glu His Asp 2074PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-N-Me-TyrMOD_RES(4)..(4)Asp-aldehyde 7Tyr
Val Ala Asp184PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TrpMOD_RES(4)..(4)Asp-aldehyde 8Trp Glu
His Asp194PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(2)..(2)N-Me-ValMOD_RES(4)..(4)N-Me-A-
sp-aldehyde 9Tyr Val Ala Asp1104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp-aldehyde 10Tyr Val
Ala Asp1114PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp-chloromethylketone
11Tyr Val Ala Asp1124PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp-2,6-dimethylbenzoyloxymet-
hylketone 12Tyr Val Ala Asp1134PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp(OtBu)-aldehyde-dimethyl
acetol 13Tyr Val Ala Asp1144PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp-aldehyde 14Tyr Val
Lys Asp1154PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(3)..(3)Lys(biotinyl)MOD_RES(4)..(4)A-
sp-2,6-dimethylbenzoyloxymethylketone 15Tyr Val Lys
Asp1164PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
peptide"MOD_RES(1)..(1)Biotinyl-TyrMOD_RES(4)..(4)Asp-chloromethylketone
16Tyr Val Ala Asp1175PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ethoxycarbonyl-AlaMOD_RES(5)..(5)Asp-aldehyde
17Ala Tyr Val Ala Asp1 5184PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TyrMOD_RES(4)..(4)Asp-chloromethylket-
one 18Tyr Val Ala Asp1194PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TyrMOD_RES(4)..(4)DL-Asp-fluoromethyl-
ketone 19Tyr Val Ala Asp1204PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Biotin-TyrMOD_RES(4)..(4)Asp-fluoroacyloxymethylke-
tone 20Tyr Val Ala Asp1214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TyrMOD_RES(4)..(4)Asp-acyloxymethylketone
21Tyr Val Ala Asp1224PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TyrMOD_RES(4)..(4)Asp-fluoromethylket-
one 22Tyr Val Ala Asp1235PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-ValMOD_RES(5)..(5)Asp-aldehyde 23Val Asp
Val Ala Asp1 5245PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-ValMOD_RES(5)..(5)Asp-fluoromethylket-
one 24Val Asp Val Ala Asp1 5254PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-GluMOD_RES(4)..(4)Asp-aldehyde 25Glu Ser
Met Asp1264PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-IleMOD_RES(4)..(4)Asp-aldehyde 26Ile Glu
Thr Asp1274PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-aldehyde 27Asp Glu
Val Asp1284PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-aldehyde 28Asp Met
Gln Asp1294PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Biotinyl-AspMOD_RES(4)..(4)Asp-aldehyde
29Asp Glu Val Asp1304PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-AspMOD_RES(4)..(4)Asp-chloromethylket-
one 30Asp Glu Val Asp1314PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-Asp(OMe)MOD_RES(2)..(2)Glu(OMe)MOD_RE-
S(4)..(4)DL-Asp(OMe)-fluoromethylketone 31Asp Glu Val
Asp1325PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
peptide"misc_feature(1)..(1)/note="Biotinylated"MOD_RES(1)..(1)Any
amino acidMOD_RES(5)..(5)Asp-fluoromethylketone 32Xaa Asp Glu Val
Asp1 5334PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-chloromethylketone
33Asp Glu Val Asp1344PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)N-benzyloxycarbonal-Asp(OMe)MOD_RES(2)..(2)Glu(OMe-
)MOD_RES(4)..(4)Asp(Ome)-fluoromethylketone 34Asp Glu Val
Asp1354PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-LeuMOD_RES(4)..(4)Asp-aldehyde 35Leu Glu
Val Asp1364PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TyrMOD_RES(4)..(4)DL-Asp-fluoromethyl-
ketone 36Tyr Val Ala Asp1374PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TrpMOD_RES(4)..(4)Asp-fluoromethylket-
one 37Trp His Glu Asp1384PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TrpMOD_RES(4)..(4)Asp-aldehyde 38Trp Glu
His Asp1394PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-TrpMOD_RES(2)..(2)Glu(O-Me)MOD_RES(4)-
..(4)Asp(O-Me) fluoromethylketone 39Trp Glu His
Asp1404PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-ValMOD_RES(4)..(4)Asp-aldehyde 40Val Glu
Ile Asp1414PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-ValMOD_RES(4)..(4)Asp-fluoromethylket-
one 41Val Glu Ile Asp1424PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-Asp(OMe)MOD_RES(4)..(4)Asp(OMe)
fluoromethylketone 42Asp Gln Met Asp1434PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-aldehyde 43Asp Glu
Val Asp1444PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Biotin-AspMOD_RES(4)..(4)Asp-fluoromethylketone
44Asp Glu Val Asp1454PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-AspMOD_RES(4)..(4)Asp-fluoromethylket-
one 45Asp Glu Val Asp1464PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-aldehyde 46Asp Glu
Val Asp1474PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-IleMOD_RES(4)..(4)Asp-aldehyde 47Ile Glu
Pro Asp1484PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-IleMOD_RES(4)..(4)Asp-aldehyde 48Ile Glu
Thr Asp1494PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-TrpMOD_RES(4)..(4)Asp-aldehyde 49Trp Glu
His Asp1504PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Boc-AlaMOD_RES(4)..(4)Asp-aldehyde 50Ala Glu
Val Asp1514PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-IleMOD_RES(4)..(4)Asp-fluoromethylket-
one 51Ile Glu Thr Asp1524PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-AspMOD_RES(4)..(4)Asp-aldehyde 52Asp Glu
Val Asp1534PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-LeuMOD_RES(4)..(4)Asp-aldehyde 53Leu Glu
His Asp1544PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Ac-LeuMOD_RES(4)..(4)Asp-chloromethylketone
54Leu Glu His Asp1554PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
peptide"MOD_RES(1)..(1)Carbobenzoxy-LeuMOD_RES(4)..(4)Asp-fluoromethylket-
one 55Leu Glu His Asp15621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 56agcacaggac acatcagttg g 215722DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 57cttgaggcag tagttcttgg tg 22
* * * * *